Comparison of human lymphotoxin gene expression in CHO cells directed by genomic DNA or cDNA sequences. Four recombinant plasmids coding for human lymphotoxin (LT) were constructed with genomic DNA (gDNA) or cDNA sequences. The simian virus 40 (SV40) early region, which contains the early promoter, an intron of the small-t-antigen-encoding gene, and polyadenylation signal sequences, was used for transcriptional and post-transcriptional regulatory elements in the construction of these plasmids. Two of them contained gDNA and the other two contained cDNA. One of the gDNA plasmids and one of the cDNA plasmids carry the SV40 intron between the structural gene and polyadenylation signal. Transient and stable gene expression levels of these plasmids in Chinese hamster ovary (CHO) cells were measured by assaying the secreted LT. The plasmid carrying gDNA without the SV40 intron was expressed more efficiently than the other three plasmids in both transient and stable gene expression assays.


Features of regenerated clones with or without fusion treatment between auxotrophic mutants of Streptomyces antibioticus and their antibiotic productivity. During experiments on protoplast fusion of complementary auxotrophic mutants (194 and 11M-21) of Streptomyces antibioticus for strain improvement, the clones (typified by F-40) regenerated on minimal regeneration medium (MRM) were found to be prototrophs, and to produce an antibiotic different from those produced by the parent strain. The protoplast regeneration of each parent was examined as a negative control experiment. In the regenerated clones of 194, half of them produced actinomycins similar to those produced by the original mutant 194, but others (typified by R-20) seemed to produce antibiotics similar to those produced by F-40. In the taxonomic characterization of morphological, cultural, and physiological properties of each strain, F-40, R-20, and the parent mutant 194 had no significant differences with a few exceptions. The problem here is whether the antibiotic of R-20 is the same as that of F-40, which was first isolated and found to be a peptide antibiotic different from actinomycins, with activity against Gram-negative and Gram-positive bacteria.


An epitope on membrane-bound but not secreted IgE: implications in isotype-specific regulation. Immunoglobulins (Igs) on the surface of B lymphocytes are isotype-specific immunological markers of the B-cell subsets expressing them. Since these membrane-bound Igs (mIgs) are antigen receptors, their interaction with antibodies could be explored for modulating the activity of specific B-cell subsets. Targeting mIgs by antibodies in vivo, however, has not been feasible because of the presence of Igs in the circulation and the frequent association of Igs with various cell types via Fc receptors. To circumvent these problems, we proposed that the extracellular portions of the membrane-anchoring segments of the heavy chains of mIgs, referred to as "mIg isotype-specific" or "migis" peptides, may provide the antigenic sites for the isotype-specific targeting of B cells in vivo. Here we describe the exemplary development of monoclonal antibodies (mAbs) recognizing this unique epitope of mIgE.


A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis. We present a simple and flexible procedure to elicit and assay anti-peptide antibodies without peptide synthesis. It consists of expressing the peptide of interest in the form of a genetic insert within two different "recipient" bacterial proteins. One hybrid protein is used as immunogen for the induction of antibodies against the inserted peptide and the other as antigen for monitoring the anti-peptide antibodies raised. The two "recipient" proteins used are the MalE and the LamB proteins from E. coli. The MalE hybrid proteins can be affinity purified on an amylose column using mild nondenaturing conditions and can be crystalized for structural studies; LamB hybrid proteins express the inserted peptide on the cell surface so that intact bacteria can be used as a reagent. We chose, as a model peptide, a B-cell epitope from the pre-S(2) region of Hepatitis B virus. With both MalE and LamB hybrid proteins, high titres of anti-preS antibodies, able to react with native HBsAg particles, were induced in mice. The anti-peptide antibody titres recorded by ELISA were comparable to those obtained when either a synthetic peptide, or the hybrid proteins, were used as immobilized antigen.


Characterization of a novel NTP-dependent 3' exoribonuclease from yeast mitochondria. We have purified and characterized a novel exoribonuclease that was isolated from the mitochondria of Saccharomyces cerevisiae. The enzyme degraded RNA in a 3' to 5' direction and was dependent on nucleotide triphosphates for activity. All eight of the standard ribo- and deoxyribonucleotide triphosphates supported activity with an apparent Km ranging from 20 to 90 uM. The enzyme also exhibited an RNA-dependent ATPase activity. Evidence suggests that in vivo the enzyme may associate with mitochondrial factors which can alleviate the dependence on nucleotide triphosphates for enzymatic activity. A model is discussed for the role of the enzyme in regulating the turnover of mitochondrial RNAs.


Epitope mapping in Salmonella flagellar protein. The flagellar filaments of bacteria of the genus Salmonella are highly immunopotent and antigenically diverse. It is proposed to develop vaccines by replacing the flagella of live attenuated Salmonella strains with engineered flagellar filament proteins containing foreign epitopes of medical and agricultural importance. As an initial step in this process, the major linear epitope regions of one filament protein have been identified.


Effect of cytokines on bovine mammary gland immunity. Cytokines are a family of glycoproteins produced by various cell types in response to specific stimuli that regulate the immune response. This paper reviews recent studies on two different cytokines, each with its own effector cell type: interleukin-2 (IL-2), which regulates lymphoid cell responses; and granulocyte colony-stimulating factor (GCSF), which regulates neutrophil responses. In the first study, administration of IL-2 to the bovine mammary gland was found to stimulate the expansion of lymphocyte populations and increase local antibody production. In the second study, systemic administration of GCSF increased peripheral blood as well as milk neutrophil populations, which afforded some protection against Staphylococcus aureus challenge. Results suggest a role for cytokines in the control of mastitis in dairy cattle.


Protein overproduction in Escherichia coli: RNA stabilization, cell disruption and recovery with a cross-flow microfiltration membrane. After optimizing overproduction of a heterologous gene product (chloramphenicol acetyltransferase, CAT) using an RNA stabilization vector * in Escherichia coli (Chan et al., 1988), a single step cell disruption and recovery method * for obtaining a product stream essentially free of cell debris was developed. The behavior of an RNA stabilization plasmid (pKTN-CAT) containing stabilizing intron RNA was investigated in two different media both in batch and chemostat modes. CAT production of pKTN-CAT was consistently higher (3- to 7-fold) than that of the control lacking the stabilization sequences (pK-CAT). Highest CAT production was observed for cells grown in minimal medium in batch mode and induced for CAT expression early in growth. CAT production of cells grown in the chemostat mode exhibited an optimal dilution rate of about 0.1 h-1. Enhancement of protein production by pKTN-CAT as compared to pK-CAT tended to be higher when grown in rich medium rather than in minimal medium. Presence of the RNA stabilization plasmid did not significantly alter the growth rate of the cell. Using a combination of chemical treatment (1 mM EDTA) and shear stress resulting from cross-flow in a stainless steel microfiltration membrane *, CAT was released into the medium through disruption of the E. coli cells. The permeate flux increased from 2000 to 9000 kg m-2 h-1 with increasing axial Reynolds number from 10,000 to 60,000 or increasing mean shear stress from 12 to 47 Pa. The turbidity of the permeate was approximately 4% that of the retentate over this range of axial flow rates, indicating excellent removal of cell debris. Also, the concentration of CAT in the permeate was equal to that in the retentate over this range of axial flow rates, indicating complete passage of protein through the membrane. Thus, using a combination of chemical treatment and fluid-induced shear stress in a cross-flow membrane module, we were able to disrupt and recover the heterologous protein in a stream low in debris.


Xyloglucosides of benzyl and phenethyl alcohols and Z-hex-3-en-1-ol from leaves of Alangium platanifolium var. trilobum. From a methanolic extract of leaves of Alangium platanifolium var. trilobum, benzyl alcohol xylopyranosyl(1---6)glucopyranoside, phenethyl alcohol xylopyranosyl(1----6)glucopyranoside and Z-hex-3-en-1-ol (aoba alcohol) xylopyranosyl(1----6)glucopyranoside were isolated. Their structures were determined mainly by NMR spectroscopy.


Predicting antigenic sites on proteins.

Predicting antigenic sites on proteins. The ability to predict antigenic sites on proteins is of major importance for the production of synthetic peptide vaccines and synthetic peptide probes of antibody structure. Many predictive methods, based on various assumptions about the nature of the antigenic response have been proposed and tested. This review will discuss the principles underlying the various approaches to predicting antigenic sites and will attempt to answer the question of how well they work.


Application of a nuclease from rye nucleus for structural studies of plant ribonucleic acids. A new nuclease (Rn) isolated from rye nucleus was applied for the structural studies of methionine initiator transfer ribonucleic acid and ribosomal 5S rRNA from yellow lupin seeds. The enzyme shows high specificity for some regions of both RNAs. The dihydrouridine and ribothymidine loops which are supposed to be involved in the tertiary interactions of the methionine initiator tRNA were hydrolysed. The anticodon loop is not digested at all. 5S rRNA was digested in single stranded regions (loops). The cleavage pattern of the tRNA and 5S rRNA obtained with Rn enzyme, suggests not only the high specificity toward single stranded regions, but also some dependence on their tertiary structure.


Towards third-generation whooping cough vaccines. To date, the most significant use of recombinant-DNA technologies has been to hyperproduce natural molecules that are difficult to obtain in large quantities by conventional methods. However, genetic manipulation can also be an efficient way to modify the properties of natural molecules in order to make them more suitable for human use. In the development of third-generation whooping cough vaccines, recombinant-DNA methods were used to remove the enzymatic activity of pertussis toxin in order to obtain a new molecule which is devoid of toxicity, and can be used for safer vaccination against this disease.


Common principles in protein folding and antigen presentation. The regular recurrence of hydrophobic amino acid residues along a peptide sequence determines the formation of a longitudinal hydrophobic strip when the peptide forms an alpha-helix. An understanding of the ways this may affect both folding of nascent proteins and antigen presentation should facilitate vaccine and therapeutics design.


Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies. Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.


Purification of glycoproteins by selective transport using concanavalin-mediated reverse micellar extraction. A novel methodology for coupling liquid-liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar-binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT-isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin-glycoprotein complex (96,000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water-pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.


Analysis of polymer molecular weight distributions in aqueous two-phase systems. The partitioning of proteins and other biomaterials between two aqueous phases containing polyethyleneglycol and dextran is a strong function of the molecular weight of the two polymers. Although both polymers are polydispersed (especially Dx) most theoretical treatments refer only to the average molecular weight (number or mass) and assume that the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distributions of four stock solutions of PEG (4000, 6000, 10,000 and 20,000) and four stock solutions of Dx (10,000, 40,000, 110,000 and 500,000) were measured using High Performance Gel Chromatography. The measurements were repeated on the phases formed by the polymer solutions after they were mixed and allowed to equilibrate. The molecular weight distribution of the Dx differed in the top and bottom phase; both differed from that of the stock solution. Although we believe that the molecular weight distribution for PEG also differs in the top and bottom phases, we were unable to determine this within the resolution of our instruments.


Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth. Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm3 per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed.


Protein isolation by solution-controlled gel sorption. Illustrated are the principles for isolating proteins from solution by sorption into a polymer gel phase, driven by the addition of a water-soluble polymer to the protein solution. The separation is shown to be analogous to conventional two-phase aqueous extraction. However, the use of a gel phase rather than a solution for absorbing the protein makes separation of the protein from the polymer and the recycling of the gel phase much simpler. The model system used was linear poly(ethylene glycol) (PEG) and dextran gel. Increasing the molecular weight and concentration of the PEG favored sorption by the gel of ovalbumin, bovine serum albumin, cytochrome c, and hemoglobin. The proteins could be quantitatively recovered by immersing the gel in PEG-free solution.


The linear PCR reaction: a simple and robust method for sequencing amplified rRNA genes. The linear polymerase chain reaction was used to sequence amplified RNA genes from strains of Bacillus, Thermus and Legionella. The technique described is simple and reproducible and it works well with double standard product which has been PEG precipitated directly from PCR reactions.


Nine phenethyl alcohol glycosides from Stachys sieboldii. Three new phenethyl alcohol glycosides together with six known compounds have been isolated from the leaves of Stachys sieboldii. On the basis of chemical and spectral analyses, the structures of three new compounds named stachysosides A, B and C have been established as 2-(3,4-dihydroxyphenyl)ethyl O-alpha-L-arabinopyranosyl-(1----2)-alpha-L-rhamnopyranosyl- (1----3)-4-O-E-caffeoyl-beta-D-glucopyranoside, 2-(3,4-dihydroxyphenyl)ethyl O-alpha-L-arabinopyranosyl-(1----2)-alpha-L-rhamnopyranosyl- (1----3)-4-O-E-feruloyl-beta-D-glucopyranoside and 2-(3-hydroxy-4-methoxyphenyl)ethyl O-alpha-L-arabinopyranosyl-(1----2)-alpha-L-rhamnopyranosyl- (1----3)-4-O-E- feruloyl-beta-D-glucopyranoside, respectively.


Retrotransposon gene engineering.

Retrotransposon gene engineering. We have used a mobile mouse VL30 genetic element together with retroviral helper cells to efficiently transmit and express chimeric foreign gene sequences in murine and human cells. The construct comprised a cDNA copy of retrotransposon NVL3, an internal promoter [rat cytosolic phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32)] and an expressed bacterial neomycin resistance gene. Thirty to sixty thousand colony forming units/ml (CFU/ml) were recovered from the supernatant of mass cultured psi2 helper cells transfected with the recombinant retrotransposon plasmid DNA. RNA was expressed from both the VL30 long terminal repeat and from the internal PEPCK promoter, resulting in a G418 drug resistance phenotype in recipient cells. Integrated VL30 DNA sequences transduced from psi2 or PA317 retroviral helper cells failed to regenerate detectable replication competent virus. Human and rodent recipient cells transduced by the retrotransposons appeared to bear intact vector sequences after two rounds of transmission by helper cells.


Inhibitors of angiogenesis.

Inhibitors of angiogenesis. Angiogenesis, the formation of new capillaries, is essential to a number of important physiological events, both normal and pathological. Recently, increased attention has focused on the purification and characterization of inhibitors of this process, because of the potential therapeutic value of angiogenesis inhibitors in controlling such "angiogenic diseases" as proliferative retinopathy, solid tumors, rheumatoid arthritis, and neovascular glaucoma. We review the process of neovascularization and the assays that have been developed to study its inhibition in vivo and in vitro. We also discuss the properties of different angiogenesis inhibitors and examine the mechanisms by which such inhibitors could potentially intervene in the process of neovascularization.


Inhibitory effect of new imidazole derivatives on the proliferation and nucleic acid synthesis of leukemic cells. New derivatives of imidazothiazole and imidazobenzothiazole were tested in vitro for their potential antiproliferative activity. Four imidazobenzothiazole derivatives exhibited a cytotoxic activity against two leukemic cell lines, compound I being the most effective. Cell cycle kinetics studies showed that this drug delays the progression of cells from G1 to S and G2 M phases. An inhibitory effect on DNA and RNA synthesis was also observed. The antiproliferative effect of this compound, analogue of immunosuppressive agents, suggested that it could be of interest for a therapeutic use and for the synthesis of new derivatives.


Polymer fractionation in aqueous two-phase polymer systems. We consider the effects of the addition of poly(ethylene glycol) (PEG) of different molecular weights to aqueous two-phase system of PEG 8000 and dextran 500. The first purpose of this study was to determine the molecular weight partitioning of the polymers themselves so that, for example, aqueous two-phase separations using affinity ligands can be improved. The second purpose was to examine whether this molecular weight partitioning could be predicted by using solution thermodynamic models so that it would be possible to optimize affinity partitioning without extensive laboratory work. Experimentally, we find that, by increasing the PEG concentration of any molecular weight in the feed, the high molecular weight PEG concentration in the dextran-rich phase is reduced. This observation can be used to reduce the loss of expensive ligated PEG used in affinity partitioning. Further, there is generally good agreement between our experimental data and the predictions of a solution thermodynamic model.


Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered Namalwa KJM-1 cells. An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 micrograms/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7 x 10(5) cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 4 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1 x 10(7) cells/ml), and the amount of G-CSF reached 41 micrograms/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.


Effect of shear stress on cytosolic Ca2+ of calf pulmonary artery endothelial cells. The purpose of the present study was to determine if hemodynamic shear stress increases free cytosolic Ca2+ concentration ([Ca2+]i) of cultured pulmonary artery endothelial cells exposed to steady laminar fluid flow in a parallel plate chamber. Average [Ca2+]i was estimated by measuring cell-associated fura-2 fluorescence using microfluorimetric analysis. To determine [Ca2+]i close to the membrane surface, 86Rb+ efflux via Ca(2+)-dependent K+ channels was measured. Upon initiation of flow or upon step increases in flow, no change in [Ca2+]i was observed using fura-2. However, increases in shear stress produced a large, transient increase in 86Rb+ efflux. The shear stress-dependent increase in 86Rb+ efflux was not blocked by either tetrabutylammonium ions (20 mM) or by charybdotoxin (10 nM), two specific inhibitors of the Ca(2+)-dependent K+ channel of vascular endothelial cells. These results demonstrate that shear stress per se has little effect on either the average cytosolic [Ca2+]i as measured by fura-2 or on [Ca2+]i close to the cytoplasmic surface of the plasmalemma as measured by the activity of Ca(2+)-dependent K+ channels.


Identification of the phenotypic modulation of rabbit arterial smooth muscle cells in primary culture by flow cytometry. In atherosclerotic lesions, smooth muscle cells (SMC) change from a contractile to a synthetic phenotype. The in vivo and in vitro phenotypic transformations of SMC have been confirmed by transmission electron microscopy (TEM), but the relationship between this change and the cell cycle is still unknown. We demonstrated the structural modulation of rabbit arterial SMC in primary culture by TEM and immunocytochemistry and simultaneously studied changes in two-dimensional histograms of the relative DNA and RNA contents by flow cytometry. During the first day of primary culture, the cells exhibited the contractile phenotype and were composed of a population in the G0 phase characterized by low contents of DNA and RNA. On the second day of culture, some of the cells (18.2%) had started but not completed the transition into the synthetic phenotype and a cell population in the G1A phase with an RNA content above the G0 level appeared in almost the same proportion. This cell population could be categorized as an "intermediate" type. Moreover, after 3 days when about three-quarters of the cells had undergone structural transition, the same proportion of cells had entered into the cycling phase, while some cells still remained in the G0 and G1A phases. Thus, cell cycle analysis by flow cytometry corresponded well with the observations obtained by TEM and immunocytochemistry. These results show that flow cytometry can rapidly and relatively conveniently monitor the process of phenotypic modulation in SMC and is a useful method for the analysis of such transitions.


Adhesion to thrombospondin by human embryonic fibroblasts is mediated by multiple receptors and includes a role for glycoprotein 88 (CD36). Fetal embryonic fibroblasts attach and spread on thrombospondin (TSP). Adhesion is tight and focal adhesion plaques and "spots" are formed. We have investigated the receptors responsible for this adhesion. Unstimulated cells express the vitronectin receptor on their surface and this beta 3 integrin molecule contributes to adhesion. Another putative receptor for TSP, termed glycoprotein (GP) 88, which exists as a cytoplasmic pool in unstimulated cells becomes surface expressed when these cells are plated on TSP and localizes to areas of cell adhesion. Western blot analysis of cell lysate confirms GP88 as a TSP binding protein. Studies with fucoidan indicate that the heparan sulfate proteoglycan, known to function as a receptor for TSP, appears to contribute substantially to the TSP attachment of these cells and may be the receptor most important in the initial phases of TSP interaction.


DNA/RNA synthesis and labelling.

DNA/RNA synthesis and labelling. Reliable methods of machine-aided RNA synthesis have been established to complement those for DNA assembly. Oligonucleotides containing thio-modified backbones and 2'-O-alkyl sugars head the list of many newly available analogues. Biotin, fluorescent agents and many reporter groups can be conveniently introduced into oligonucleotides in multiples by phosphoramidite or H-phosphonate chemistry.


Surface presentation of protein epitopes using bacteriophage expression systems. Vast libraries of filamentous phage expression vectors that display foreign (poly)peptides on the virion surface can be screened by affinity-purifying those phage whose displayed foreign peptide binds to an antibody or another binding protein. Present libraries display only short random peptides, but work is presently underway to create libraries displaying antibodies with a great diversity of binding specificities.


Brain metastases and testicular tumors: long-term survival. In this updated and expanded retrospective analysis, the treatment records of 24 patients with brain metastases from nonseminomatous germ cell testicular tumors (NSGCT's) treated at the Indiana University Department of Radiation Oncology from 1975 through 1988 were reviewed. All patients received standard cisplatin-based induction chemotherapy. These patients were divided into three groups. Group 1 (n = 10) consisted of patients who presented initially with brain metastases and had no prior systemic treatment. Group 2 (n = 4) consisted of those patients who, after achieving a complete response (CR) with cisplatin, vinblastine, and bleomycin (PVB) +/- doxorubicin, developed a relapse confined to the brain. Group 3 (n = 10) consisted of those patients who were initially treated with PVB +/- doxorubicin or bleomycin, etoposide, and cisplatin (BEP) and eventually developed progressive disease and brain metastases. Group 1 was treated with whole brain irradiation (WBRT) and PVB +/- doxorubicin or BEP. Group 2 was treated with WBRT, cisplatin-based chemotherapy +/- surgical excision. Group 3 was usually treated with WBRT palliatively. Six patients, three in Group 1 and three in Group 2, are alive and disease-free with follow-up of 5+ years from beginning WBRT. Two additional patients in Group 1 survived 5+ years from beginning WBRT before dying with disease. No patient in Group 3 survived. Patients with brain metastases who have potentially controllable systemic disease should be treated curatively with WBRT (5000 cGy/25 fractions) +/- surgical excision and concomitant chemotherapy.


Changes in brain monoaminergic neurotransmitter concentrations in rat after intracerebroventricular injection of streptozotocin. The tissue concentrations of the monoaminergic neurotransmitters noradrenaline (NA), dopamine, and serotonin (5-HT) and of their major metabolites were measured by HPLC and electrochemical detection in several rat brain areas after intracerebroventricular injection of streptozotocin (STZ). NA levels were found to be decreased in the frontal cortex by 14%, in the entorhinal cortex by 18%, and in the striatum by 38%. In the entorhinal cortex, 5-HT levels were decreased by 19% and the 5-HT turnover rate, measured as the 5-hydroxyindoleacetic acid/5-HT ratio, was found to be increased by 48%. These results may be indicative of a distinct susceptibility of some neurotransmitters in certain brain areas after a more general impairment of brain metabolism by means of intracerebroventricular application of the diabetogenic compound STZ.


Exercise and the brain: angiogenesis in the adult rat cerebellum after vigorous physical activity and motor skill learning. This study compared the morphology of cerebellar cortex in adult female rats exposed for 1 month to repetitive exercise, motor learning, or an inactive condition. In the exercise conditions, rats that were run on a treadmill or housed with access to a running wheel had a shorter diffusion distance from blood vessels in the molecular layer of the paramedian lobule when compared to rats housed individually or rats that participated in a motor skill learning task. Rats taught complex motor skills substantially increased the volume of the molecular layer per Purkinje neuron and increased blood vessel number sufficiently to maintain the diffusion distance. These results dissociate angiogenesis associated with increased neuropil volume (as seen in the motor learning group) from angiogenesis associated with increased metabolic demands (as seen in the exercise groups). While the volume fraction of mitochondria did not differ among groups, the mitochondrial volume fraction per Purkinje cell was significantly increased in the motor skill rats. This appears to parallel the previously reported increase in synapses and associated neuropil volume change.


Effects of inhibitors of protein kinase C and calpain in experimental delayed cerebral vasospasm. Vasospasm was produced in adult mongrel dogs by a two-hemorrhage method, and the spastic basilar arteries were exposed via the transclival route on Day 7. Tonic contraction was produced in the normal canine basilar arteries by a local application of KCl or serotonin after transclival exposure. The exposed spastic and tonic basilar arteries then received a topical application of the following: 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7), a potent inhibitor of protein kinase C acting at the catalytic domain; calphostin C, a specific inhibitor of protein kinase C acting at the regulatory domain; or calpeptin, a selective inhibitor of calpain. Both spastic and tonic basilar arteries were effectively dilated by H-7. Calphostin C caused only slight dilation of spastic basilar arteries but moderate dilation of tonic basilar arteries. Dilation in response to calpeptin was remarkable in the spastic basilar arteries but slight in the tonic basilar arteries. The doses of calphostin C and calpeptin required to obtain maximum effect were markedly lower in the tonic model than in the spastic model. The spastic and tonic models had a similar dose-dependent response to H-7 but quite a different response to calphostin C or calpeptin, suggesting a difference in the function of protein kinase C and calpain in the two models. Furthermore, the effect of calphostin C on the reversal of vasospasm was increased significantly after topical treatment with calpeptin. It is suggested that the majority of the catalytic domain of protein kinase C is dissociated from the regulatory domain, probably by a limited proteolysis with calpain, and is markedly activated in vasospasm.


Humoral hypercalcemia in seminomas.

Humoral hypercalcemia in seminomas. Seminomas are germ cell tumors that are rarely associated with hypercalcemia. In this report, four cases of seminoma with concomitant hypercalcemia are presented and another three from the literature are reviewed. All seven patients exhibited hypercalcemia with a normal serum concentration of inorganic phosphorus and no evidence of skeletal metastases. The peripheral venous level of parathyroid hormone (PTH) was normal in four of the five patients in whom it was measured. The serum concentration of calcitriol was elevated in the two patients in whom it was measured. After systemic chemotherapy, the serum "corrected" total calcium concentration returned to normal and remained normal; the decrease in the levels temporally paralleled the decrease in tumor volume. Both patients with elevated calcitriol levels remained eucalcemic after treatment of the malignancy, suggesting that the increased serum calcitriol level was linked to the development of hypercalcemia as this humoral agent was inappropriately elevated by patients with this syndrome. In contrast to many forms of malignancy, the development of hypercalcemia did not adversely affect the prognosis of the patients with seminoma, since all seven patients entered complete remission. Hypercalcemia appears to be heretofore unrecognized paraneoplastic syndrome associated with seminoma.


Isolated central nervous system relapse of testicular cancer. Central nervous system involvement with testicular cancer usually occurs with advanced systemic disease. Isolated CNS disease at relapse is rare. We report a patient who developed a solitary brain metastasis with no other systemic disease after having achieved a complete response to frontline therapy. After combined modality therapy for the CNS disease, the patient has remained disease-free for more than 3 years. The literature regarding brain metastases in relapsed testicular cancer is reviewed, including nine cases of isolated brain metastases. The CNS can be a "sanctuary" site for testicular cancer, and in the unusual subset of patients with isolated brain relapse, long-term remission is possible with aggressive therapy.


Peripheral neovascularization of muscle and musculocutaneous flaps in the pig. Late loss of free muscle flaps following surgical or accidental trauma to the dominant vascular pedicle has been reported. In this study, time-dependent ligation of the dominant vascular pedicle was undertaken in denervated latissimus dorsi musculocutaneous or muscle-only island flaps in the pig. Muscle flaps were covered with a skin graft, and silicon rubber sheets were inserted between the flaps and their bases to simulate a poorly vascularized bed. Hemodynamic and viability studies were then performed using intravenous fluorescein (skin viability), tetrazolium blue (muscle viability), and radiolabeled 15-micron microspheres (capillary blood flow). Blood flow did not change in acutely raised musculocutaneous flaps (n = 10) but was significantly elevated in acutely raised muscle-only flaps (n = 10), suggesting that the skin paddle may steal blood flow from the underlying muscle in musculocutaneous flaps. Peripheral neovascularization at 1 day to 8 weeks was assessed (n = 30). Viability increased during the first week of revascularization and was not different in musculocutaneous and muscle-only flaps. Revascularization of muscle-only flaps was enhanced compared with musculocutaneous flaps in the 2- to 8-week period.


Sclerotherapy of malignant pleural effusion through sonographically placed small-bore catheters. Pleural sclerosis after drainage with a small-bore catheter was performed in 21 patients with malignant pleural effusions. Intrapleural catheters 7- to 24-French in size were placed by using sonographic guidance. Tetracycline (18 patients) and bleomycin (four patients) were used as sclerosing agents (one patient had both). Clinical and radiologic follow-up was available on all patients until they died (range, 2 weeks to 25 months; mean, 3.6 months). Pleural sclerosis was successful in 15 (71%) of 21 patients. Two patients in whom pleurodesis failed had pleural sclerosis repeated, with one success and one failure. All of the failures were in patients in whom the amount of chest-tube drainage was more than 100 ml/day. Pleurodesis with tetracycline was painful in six patients; no pain was associated with use of bleomycin. Small pneumothoraces developed in four patients at the time of chest-tube placement, without consequence. A superimposed infection that developed in a patient having continuous drainage of pleural fluid was successfully treated with antibiotics. Pleural sclerotherapy can be performed through sonographically placed small-bore catheters with results comparable to those seen with large-bore, surgically placed catheters.


Increased sensitivity of human keratinocytes immortalized by human papillomavirus type 16 DNA to growth control by retinoids. Human papillomavirus (HPV) type 16 (HPV16) is associated with a large percentage of cervical malignancies, and HPV16 DNA can immortalize human keratinocytes in vitro. The transforming ability of the virus resides primarily in the open reading frames E6 and E7. Retinoids are potent modulators of growth and differentiation of keratinocytes and have been shown to reverse cervical lesions resulting from HPV infection. We compared the sensitivity of normal human foreskin keratinocytes (HKc) and four immortalized HKc lines, independently obtained by transfection of different normal HKc strains with HPV16 DNA (HKc/HPV16), to growth control by retinoic acid (RA). All the HKc/HPV16 lines were 10- to 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc, while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. In addition, HKc/HPV16 lines were more sensitive than normal HKc to modulation of keratin expression by RA and retinol. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Dot blot analysis of RNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 open reading frames E6 and E7 is reduced 2- to 4-fold by RA. In addition, Northern blot analysis demonstrated that RA inhibition of E6 and E7 expression was both dose and time dependent. Overall, these results suggest that the increased sensitivity of the HKc/HPV16 lines to growth control by RA may be mediated by an inhibition of the expression of HPV16 gene products which are required for the maintenance of continuous growth.


Intracellular metabolism of 5-formyl tetrahydrofolate in human breast and colon cell lines. This report describes the intracellular metabolism of 5-formyltetrahydrofolate into the various one-carbon substituted folate and polyglutamate pools in a human breast (MCF-7) and colon (HCT 116) carcinoma cell line. Metabolism into the one-carbon substituted pools was found to be time and dose dependent over a concentration range up to 50 microM. A 3-fold increase in total intracellular folate was noted over a 50-fold concentration range (1-50 microM) of 5-formyltetrahydrofolate tested in the colon cell line, while in the breast line, a 6-fold increase was detected over a 500-fold concentration range (0.1-50 microM). The level of 5, 10-methylenetetrahydrofolate, which was detectable only in the breast cell line, was found to increase by a factor of 10 (1.8 pmol/mg to 17.9 pmol/mg) over the concentration range studied. The majority of metabolism was into the 10-formyltetrahydrofolate and tetrahydrofolate pools in the breast cells and into the 5-methyltetrahydrofolate pool in the colon cells. Polyglutamation was also time and dose dependent, with a significant proportion of the total pool represented by the higher polyglutamate forms (Glu3-Glu5) after 24 h of continuous exposure to 5-formyl tetrahydrofolate. Pentaglutamate was the highest level noted in both cell lines. The intracellular half-life of the polyglutamate forms was inversely related to the length of the polyglutamate tail with half-lives of 71, 131, 143, 441, and 1167 min for the mono- through pentaglutamate, respectively. Finally, up to a 20:1 ratio of the biologically inactive (6R) isomer to active (6S) isomer of 5-formyltetrahydrofolate resulted in no effect on metabolism into the one-carbon substituted folate pools and only minimal decreases in metabolism to the polyglutamate forms. These studies suggest that prolonged exposure to even relatively low doses of 5-formyltetrahydrofolate may be optimal for intracellular metabolism to the most biologically relevant forms for ternary complex formation with thymidylate synthase and fluorodeoxyuradylate, since longer exposures result in a greater accumulation of the higher polyglutamates.


Nitroreductases and glutathione transferases that act on 4-nitroquinoline 1-oxide and their differential induction by butylated hydroxyanisole in mice. These studies concern the initial steps in 4-nitroquinoline 1-oxide (4NQO) metabolism in relation to mechanisms of anticarcinogenesis. Butylated hydroxyanisole (BHA) administration by a protocol known to inhibit the pulmonary tumorigenicity of 4NQO in A/HeJ mice enhanced hepatic and pulmonary activities for 4NQO metabolism by two major pathways, conjugative detoxification and nitroreductive activation. High-performance liquid chromatography analysis showed approximate doubling of two types of glutathione transferase subunits with 4NQO-conjugating activity in livers of BHA-treated mice. Similar increases were observed in hepatic 4NQO-conjugating activity and in Vmax, while Km for 4NQO was 39 to 43 microM. Pulmonary 4NQO-glutathione transferase activity increased 24 to 29%. DT diaphorase activity toward 4NQO was elevated 3.3-fold in livers and 2.7-fold in lungs of BHA-treated mice. However, the predominant 4NQO reductase of liver and lung was dicumarol resistant, had a strong preference for NADH, and showed little if any response to BHA. This Mr 200,000 enzyme, partially purified from livers of Swiss mice, exhibited the stoichiometry of 2-NADH/4NQO expected for reduction of 4NQO to 4-hydroxyaminoquinoline 1-oxide. Its high affinity for 4NQO (Km, 15 microM) signified a much greater influence on 4NQO metabolism than DT diaphorase (Km, 208 microM). The dicumarol-resistant 4NQO reductase differed from several known cytosolic nitroreductases. The results suggest that protection by BHA may result from alteration of the balance between 4NQO activation and conjugation.


[3H]paroxetine binding and serotonin content of rat cortical areas, hippocampus, neostriatum, ventral mesencephalic tegmentum, and midbrain raphe nuclei region following p-chlorophenylalanine and p-chloroamphetamine treatment. The agents p-chlorophenylalanine (PCPA) and p-chloroamphetamine (PCA) deplete brain serotonin (5-HT) levels by two different mechanisms; PCPA inhibits the enzyme tryptophan hydroxylase, whereas PCA has a neurotoxic action on certain 5-HT neurons. The parameters of [3H]paroxetine binding to homogenates prepared from the cerebral cortex of rats treated with PCPA, PCA, or saline; vehicle were investigated. The tissue concentrations of 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) were also determined by HPLC in the same brain samples. After PCPA treatment, neither the maximum binding capacity (Bmax) nor the dissociation constant (KD) of [3H]paroxetine for the 5-HT uptake recognition site differed from controls despite a substantial reduction in the concentration of 5-HT and 5-HIAA. In contrast, significant changes in both the Bmax and KD values were observed in the cerebral cortex of rats treated with PCA. Furthermore, [3H]paroxetine binding and tissue concentrations of 5-HT and 5-HIAA were measured in the following different regions of the rat brain: cingulate, parietal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum; ventral mesencephalic tegmentum; and midbrain raphe nuclei region after administration of PCPA, PCA, or saline vehicle. There was an excellent correlation between regional 5-HT levels and specific [3H]paroxetine binding in control and PCA-treated rats although this correlation was lost after PCPA treatment. Under these conditions, the 5-HT innervation remains unchanged whereas the concentration of 5-HT and 5-HIAA is greatly reduced. Thus, [3H]paroxetine binding appears to provide a reliable marker of 5-HT innervation density within the mammalian CNS.


Glutamate receptor agonists stimulate nitric oxide synthase in primary cultures of cerebellar granule cells. The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.


Morphometric analysis of normal, mutant, and transgenic CNS: correlation of myelin basic protein expression to myelinogenesis. The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)


Potassium-induced depolarization of rat telencephalic synaptoneurosomes increases [3H]amino-3-hydroxy-5-methylisoxazole-4-propionate receptor binding. Potassium-induced depolarization of synaptoneurosomes prepared from rat telencephalon was found to increase [3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding to the AMPA receptor. The effect required the presence of calcium because it was blocked by the calcium chelator EGTA but was not blocked by an antagonist of the N-methyl-D-aspartate receptor, aminophosphonopentanoate. The depolarization-induced increase in [3H]AMPA binding was markedly reduced by a blocker of voltage-dependent calcium channels, verapamil. Saturation kinetic experiments revealed that the increase in [3H]AMPA binding produced by potassium depolarization was due to an increase in the affinity of the AMPA receptor. These results provide additional support for a critical role of calcium in the regulation of the AMPA receptors. The synaptoneurosome preparation might represent an interesting tool to determine the role of different calcium-dependent enzymes involved in the regulation of the AMPA receptor.


Vestibular compensation affects endogenous phosphorylation of frog brain proteins. The effect of unilateral labyrinthectomy followed by the process of vestibular compensation on the incorporation of radioactive phosphate into frog brain proteins was investigated. Phosphoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. The present data show that unilateral labyrinthectomy affects the incorporation of 32P into various frog brain proteins. In particular, the phosphorylation of a 20-kDa protein appeared enhanced during early stages of vestibular compensation (4-12 days). This 20-kDa protein was shown to be immunologically related to myelin basic protein and its phosphorylation was regulated by an endogenous calcium/calmodulin-dependent protein kinase. These data might indicate that in addition to neuronal components, components of glial origin are also involved in biochemical events that lead to functional recovery after neuronal lesions.


Odontogenic keratocysts: a clinical and histologic comparison of the parakeratin and orthokeratin variants. Four hundred forty-nine cases of odontogenic keratocyst (OKC) were separated into three histologic categories: parakeratinized, orthokeratinized, or a combination of the two types. Demographic and clinical data, such as anatomic location and recurrence, were obtained from the biopsy forms. Results showed that 86.2% of the 449 cases were parakeratinized, 12.2% were orthokeratinized, and 1.6% had features of both orthokeratin and parakeratin. There were no statistically significant differences between orthokeratinized and parakeratinized OKCs when age, race, sex, presenting symptoms, and the clinical impression were compared. The orthokeratinized OKC was more often associated with an impacted tooth (75.7%), as compared with 47.8% for the parakeratinized OKC (P = .001). Parakeratinized OKCs recurred in at least 42.6% of the cases, compared with only 2.2% for orthokeratinized OKCs. This study emphasizes the importance of distinguishing between the parakeratin and orthokeratin variants of OKC. In addition, data are presented that show the need for longer follow-up than previously documented.


The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity. The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.


Biological and biochemical activity of v-Crk chimeras containing the SH2/SH3 regions of phosphatidylinositol-specific phospholipase C-gamma and Src. The chicken CT10 virus oncogene product, P47gag-crk, contains SH2/SH3 domains that have been identified as conserved domains among proteins involved in signal transduction. We studied the functional similarity of the SH2/SH3 domains by replacing those of v-Crk with those of phosphatidylinositol-specific phospholipase C-gamma, v-Src, or c-Src. The transforming activity of v-Crk was partially retained in a mutant with a v-Src SH3 domain but not in the other mutants with heterologous SH2/SH3 domains. Mutant viruses with Crk-SH2/SH2' domains induced tyrosine phosphorylation of cellular proteins, but mutants with phosphatidylinositol-specific phospholipase C-gamma or Src SH2/SH2' domains did not. However, the mutant proteins with heterologous SH2/SH2' regions were able to weakly associate with some phosphotyrosine-containing proteins in vitro. These results indicate that in the context of the P47gag-crk structure, the requirement of Crk-SH2/SH3 is more stringent for its activity to induce cell transformation than to cause phosphorylation of cellular proteins. The substitution with heterologous sequences least perturbs the capacity to bind phosphotyrosine-containing proteins. In each case, the SH3 domain is more flexible to substitution than is the SH2 domain.


Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice. Expression of the human poliovirus receptor (PVR) in transgenic mice results in susceptibility to poliovirus infection. In the primate host, poliovirus infection is characterized by restricted tissue tropism. To determine the pattern of poliovirus tissue tropism in PVR transgenic mice, PVR gene expression and susceptibility to poliovirus infection were examined by in situ hybridization. PVR RNA is expressed in transgenic mice at high levels in neurons of the central and peripheral nervous system, developing T lymphocytes in the thymus, epithelial cells of Bowman's capsule and tubules in the kidney, alveolar cells in the lung, and endocrine cells in the adrenal cortex, and it is expressed at low levels in intestine, spleen, and skeletal muscle. After infection, poliovirus replication was detected only in neurons of the brain and spinal cord and in skeletal muscle. These results demonstrated that poliovirus tissue tropism is not governed solely by expression of the PVR gene nor by accessibility of cells to virus. Although transgenic mouse kidney tissue expressed poliovirus binding sites and was not a site of poliovirus replication, when cultivated in vitro, kidney cells developed susceptibility to infection. Identification of the changes in cultured kidney cells that permit poliovirus infection may provide information on the mechanism of poliovirus tissue tropism.


Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells. The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically. In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells. By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins. In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci. Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells. These results suggest that replication centers similar to those established by other viruses exist for AAV. These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro.


Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.


Influenza A virus transfectants with chimeric hemagglutinins containing epitopes from different subtypes. Influenza virus transfectants with chimeric hemagglutinins were constructed by using a ribonucleoprotein transfection method. Transfectants W(H1)-H2 and W(H1)-H3 contained A/WSN/33(H1N1) (WSN) hemagglutinins in which the six-amino-acid loop (contained in antigenic site B) was replaced by the corresponding structures of influenza viruses A/Japan/57(H2N2) and A/Hong Kong/8/68(H3N2) (HK), respectively. Serological analysis indicated that the W(H1)-H3 transfectant virus reacted with antibodies against both the WSN and HK viruses in hemagglutination inhibition and plaque neutralization assays. Furthermore, mice immunized with W(H1)-H3 transfectant virus produced antibodies to the WSN and HK viruses. The results demonstrate that influenza virus transfectants can be engineered to express epitopes of different subtypes on their hemagglutinins.


A glutamine residue in the membrane-associating domain of the bovine papillomavirus type 1 E5 oncoprotein mediates its binding to a transmembrane component of the vacuolar H(+)-ATPase. The 44-amino-acid E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. It is a highly hydrophobic polypeptide which dimerizes and localizes to the Golgi apparatus and endoplasmic reticulum membranes. Recent evidence suggests that E5 modulates the phosphorylation and internalization of the epidermal growth factor and colony-stimulating factor 1 receptors and constitutively activates platelet-derived growth factor receptors in C127 and FR3T3 cells. Although no direct interaction with these growth factor receptors has yet been identified, the E5 oncoprotein has been shown recently to interact with the hydrophobic 16-kDa component of the vacuolar H(+)-ATPase (16K protein) [D. J. Goldstein, M. E. Finbow, T. Andresson, P. McLean, K. Smith, V. Bubb, and R. Schlegel, Nature (London) 352:347-349, 1991]. In the current study, we have further analyzed the E5-16K protein complex by fast protein liquid chromatography and shown that each E5 dimer appears to bind two 16K proteins. In order to define the specific amino acid residues of E5 which participate in this binding, mutated E5 epitope fusion proteins were analyzed for their ability to coprecipitate 16K protein. Transformation-defective mutants containing amino acid substitutions within the short hydrophilic carboxyl-terminal domain retained the ability to associate with the 16K protein. However, E5 mutants lacking the glutamine residue in the hydrophobic domain were markedly inhibited in 16K protein binding. Most interestingly, the placement of a glutamine in several random hydrophobic sequences facilitated 16K protein binding, defining this residue as a potential binding site for the 16K protein component of the proton pump and exemplifying the critical role of hydrophilic amino acids for mediating specific interactions between transmembrane proteins.


Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope. We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.


Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen. Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL.


Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers. Sera from 17 of 18 adult volunteers challenged with a virulent serotype 1 rotavirus strain (D) were examined for prechallenge antibody levels against several well-defined rotavirus VP7 and VP4 neutralization epitopes by a competitive epitope-blocking immunoassay (EBA) in order to determine whether correlates of resistance to diarrheal illness could be identified. The presence of prechallenge serum antibody at a titer of greater than or equal to 1:20 that blocked the binding of a serotype 1 VP7-specific monoclonal antibody (designated 2C9) that maps to amino acid residue 94 in antigenic site A on the serotype 1 VP7 was significantly associated with resistance to illness or shedding (P less than 0.001) or illness and shedding (P less than 0.01) following challenge with the serotype 1 virus. In addition, an EBA antibody titer of greater than or equal to 1:20 in prechallenge serum against a serotype 3 VP7-specific epitope (defined by monoclonal antibody 954/159) that maps to amino acid 94 on the serotype 3 VP7 was also significantly associated with resistance to illness or shedding (P = 0.02), with a trend for protection against illness and shedding. A trend was also noted between the presence of EBA antibody against a cross-reactive VP4 epitope common to many human rotavirus strains, including the challenge virus, or a rhesus monkey rotavirus strain-specific VP4 antigenic site, and resistance to illness or shedding. These data confirm that the presence of serum antibody correlates with resistance to rotavirus illness or shedding but, in addition, demonstrate the association of antibody to a specific epitope with resistance to illness or shedding. These data also suggest that antigenic site A on the rotavirus VP7, composed of amino acids 87 to 96, may be involved in the formation of a major protective epitope. Further study of the role of this epitope in the development of homotypic and heterotypic immunity to rotaviruses following natural or vaccine-induced infection may be important in the development of strategies for control of rotavirus diarrheal disease.


p53 shares an antigenic determinant with proteins of 92 and 150 kilodaltons that may be involved in senescence of human cells. A panel of primary human cells and virus-transformed derivatives were tested for events that coincide with immortalization. In all primary and precrisis cells, two proteins of 92 and 150 kDa that shared an epitope with p53 were found; in most of their immortalized derivatives, however, they were absent. Expression of these proteins may be involved in senescence.


A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6. A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli. The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides. The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced. Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone.


Tunicamycin treatment of CHO cells abrogates multiple blocks to retrovirus infection, one of which is due to a secreted inhibitor. Chinese hamster ovary (CHO) cells are resistant to infection by all of the major classes of murine retroviruses and are partially resistant to infection by gibbon ape leukemia virus. Treatment of CHO cells with the glycosylation inhibitor tunicamycin rendered these cells susceptible to infection by retroviral vectors with ecotropic, xenotropic, and amphotropic host ranges and increased the titer of gibbon ape leukemia virus pseudotyped vectors 10-fold. Vectors having a polytropic host range did not infect CHO cells in the presence or absence of tunicamycin, showing that the effect of tunicamycin was specific and related to the pseudotype of the vector. We present evidence for three mechanisms of resistance to infection: lack of viral receptors on CHO cells, the presence of nonfunctional receptors which can be made functional by treatment with tunicamycin, and the secretion of a protein factor that blocks retroviral infection of CHO cells. Several criteria indicate that the secreted inhibitor is not an interferon, and secretion of this factor was not detected in several other cell lines that were examined.


Methylation of discrete sites within the enhancer region regulates the activity of the Epstein-Barr virus BamHI W promoter in Burkitt lymphoma lines. Eight of the nine viral antigens known to be expressed in in vitro Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines are downregulated in EBV-carrying Burkitt lymphomas (BL). Only EBNA1 can be detected in BL biopsies and BL-derived cell lines that maintain the representative phenotype during culture in vitro (group I BL lines). This restricted pattern of viral gene expression is accompanied by extensive EBV DNA methylation and can be reversed by treatment with the demethylating agent 5-azacytidine. Transcription of the genes encoding the six transformation-associated EBNAs can be initiated from one of two promoters located in the BamHI C and W regions, respectively, of the virus genome. We show that discrete sites within the BamHI W enhancer region are methylated in the group I BL lines Rael, Cheptage, and Elijah and become unmethylated after 5-azacytidine treatment that induces the expression of EBNA2. Demethylation correlates with activation of transcription from the BamHI W promoter as determined by S1 protection analysis. Reporter plasmids in which the W enhancer sequences were linked to the chloramphenicol acetyltransferase gene were active in untreated Rael, Cheptage, and Elijah cells, demonstrating that all of the required transcription factors are present in group I BL cells. Conversely, in vitro methylation of the enhancer sequences abolished their activity. The results suggest that methylation of control regions in the EBV genome may play a critical role for the regulation of viral gene expression in tumor cells.


Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression. We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression.


Medical management of AIDS patients. Kaposi's sarcoma. Kaposi's sarcoma (KS) presents with variable severity in the context of HIV infection. Various therapeutic approaches can be taken, and these are determined by the extent and location of KS lesions and the severity of tumor-related symptoms. New insights into the pathogenesis of KS lesions may provide innovative treatment strategies and a more rational basis for the control of this neoplasm.


Mutagenicity and chemical analysis of sequential organic extracts of airborne particulates. To obtain insight into the identity of chemicals associated with the mutagenicity of United States National Institute of Standards and Technology (NIST) Standard Reference Materials SRM 1649 (urban dust) and SRM 1650 (diesel particulate), parallel mutagenicity tests and chemical analyses were performed on dichloromethane and sequential organic extracts of these samples. SRM 1649 and 1650 were sequentially extracted with five organic solvents of increasing polarity, in order to partition mutagenic components into discrete fractions. The solvents (with associated polarity index) were as follows: (1) hexane (0.0); (2) hexane:diethyl ether 9:1 (0.29); (3) hexane:diethyl ether 1:1 (1.45); (4) diethyl ether (2.9); (5) methanol (6.6). 0.9270 g of SRM 1649, and 0.0510 g of SRM 1650 were each extracted three times with 8 ml of each of the solvents, the three aliquots were pooled, and analysed for target organics or solvent-exchanged into DMSO for mutagenicity testing in Salmonella typhimurium strains TA98 and TA100. The dichloromethane extracts of SRM 1649 and SRM 1650 contained direct-acting mutagens in Salmonella strains TA98 and TA100; SRM 1650 was significantly more potent than SRM 1649 in either strain. Addition of S9 caused a large decrease in mutagenicity of each extract, although SRM 1650 remained more potent. An interesting pattern of mutagenicity was observed for the sequential extracts of SRM 1649 and SRM 1650: the mutagenic potency of SRM 1649 extracts increased with increasing polarity of the extraction solvent while the response of the SRM 1650 extracts was the opposite. This suggests that the direct-acting mutagens in SRM 1650 are unlike those in SRM 1649. The response, though diminished, was largely unchanged when S9 was included in the test mixture. Chemical analyses on the various extracts were performed using a Hewlett-Packard model 5890 gas chromatograph equipped with a model 5970B mass selective detector (GC-MSD), and a 0.3 microns film thickness cross-linked methyl silicone capillary column (HP 1909A-101). Selected ion monitoring (SIM) methods were used to analyze for 105 target compounds including PAHs and nitro-PAHs. Chemical analysis of the dichloromethane extracts of SRM 1649 and SRM 1650 identified three main classes of compounds: polyaromatic hydrocarbons (PAH), nitro-polyaromatic hydrocarbons (NO2-PAHs) and heterocyclics. The concentration of target compounds and the proportion of nitro-PAHs and heterocyclic compounds were considerably greater in SRM 1650 than in SRM 1649, consistent with the observed differences in their mutagenic potency. However, the different responses of the dichloromethane extracts in TA98 and TA100 suggest the presence of different (unidentified) compounds.(ABSTRACT TRUNCATED AT 400 WORDS)


Standard reference materials for chemical and biological studies of complex environmental samples. Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) are often used in methods development and interlaboratory comparison studies since they are homogeneous and readily available to the scientific community. SRM 1649 (urban dust/organics), SRM 1650 (diesel particulate matter), and SRM 1597 (complex mixture of polycyclic aromatic hydrocarbons from coal tar) are three environmental samples which have been used by the scientific community for these purposes. These SRMs were originally developed to assist laboratories in validating analytical procedures for the determination of polycyclic organic compounds in complex mixtures. In addition, these SRMs have been valuable for the comparison of methodologies for bacterial bioassays and the development of bioassay-directed fractionation and bioassay-directed chemical analysis techniques. Most recently these SRMs were chosen for use as test samples in a collaborative study coordinated by the World Health Organization--International Programme on Chemical Safety. This paper provides a summary of much of the work to date (published and unpublished) on the chemical and biological characterization of these three SRMs. Information regarding the availability of other NIST SRMs that might be useful for these types of studies are provided also.


Mutagenicity studies on complex environmental mixtures: selection of solvent system for extraction. The mutagenic activities in the Salmonella/microsome assay of dichloromethane (DCM) and acetone extracts of complex environmental mixtures were compared. The particulate samples used in the IPCS collaborative study were Soxhlet-extracted twice with DCM followed by a third extraction with acetone. Compared with the mutagenic activity of the first extract, the third (acetone) extract of the urban particulate matter showed a relatively high mutagenic activity. In contrast to this the third extract of the diesel particulate matter contributed very little additional mutagenic activity. Furthermore, 10 filter samples of air particulates from a suburban airport area were collected for comparison of the extraction efficiency of DCM and acetone. Each sample was divided into two samples of identical size followed by extraction with acetone and DCM, respectively. No clear difference in the mutagenic activity of these extracts was observed in strains TA98 and TA98NR. It is concluded that for ambient air particulates (but not emission samples) acetone may extract some mutagenic compounds which are not extracted by DCM. The amount of these additional extractable compounds seems to depend on the composition of the sample. As DCM extracts are better suited for further fractionation and chemical analysis DCM is considered to be the best choice for a general solvent system for extraction of complex environmental mixtures.


Application of a semi-automated SOS chromotest for measuring genotoxicities of complex environmental mixtures containing polycyclic aromatic hydrocarbons. Soxhlet-extracted samples of standard reference materials (SRMs) 1649 (PAR1: urban dust/organics) and 1650 (PAR2: diesel particulate matter) from the U.S. Institute of Standards and Technology were tested for induction of SOS functions using a semi-automated version of the SOS chromotest with Escherichia coli PQ37. Concentrations of 10 polycyclic aromatic hydrocarbons in the extracts were determined using reversed-phase HPLC. Only the diesel particulate matter (PAR2) extracts expressed SOS induction activity, which decreased when metabolic activation was used. Mutagenic PAH compounds (e.g., chrysene) were found in higher concentrations in the PAR2 extracts than in the PAR1 extracts but this could not explain the genotoxicity while it was mainly exhibited without metabolic activation. The direct genotoxic activity of the diesel particulate matter sample PAR2 is probably caused by nitroaromatic compounds; this was also supported by parallel studies with the Ames/Salmonella assay.


Influence of the microsomal inducer and the incubation system on mutagenicity of complex mixtures. The mutagenicity of SRM 1649 and 1650 was tested in the presence of rat liver S9 mix which was induced by polychlorinated biphenyl (PCB) or by the combination of phenobarbital and 5,6-benzoflavone. The S9 mix induced by PCB activated benzo[a]pyrene strongly. The S9 mix induced by phenobarbital-5,6-benzoflavone activated the complex mixtures to approximately the same extent as that induced by PCB. This finding indicates that phenobarbital-5,6-benzoflavone instead of PCB may be suitable as an inducer under some conditions. The preincubation procedure for the mutagenicity test was performed by preincubating the test compound, S9 mix and bacteria for 20 min in a water bath. This procedure was as effective as the plate incorporation test.


Design and implementation of a collaborative study of the mutagenicity of complex mixtures in Salmonella typhimurium. In 1987, the International Programme on Chemical Safety (IPCS) in collaboration with the U.S. Environmental Protection Agency (U.S. EPA) and the U.S. National Institute of Standards and Technology (U.S. NIST) initiated an international collaborative study of the mutagenicity of complex environmental mixtures in the Ames Salmonella typhimurium mutation assay. The objectives of this study were: (1) to estimate the inter- and intra-laboratory variability associated with the extraction of mixtures for bioassay, (2) to estimate the inter- and intra-laboratory variability associated with the Salmonella typhimurium bioassay when applied to complex mixtures, and (3) to determine whether standard reference complex mixtures would be useful in mutagenicity studies and to evaluate whether reference or certified mutagenicity values determined from this collaborative study should be reported. The complex mixtures used in this study were selected from standard reference materials (SRMs) which had previously been issued by the U.S. NIST as SRM 1597 (coal tar), SRM 1649 (diesel particulate matter) and SRM 1650 (urban air particulate matter) with certified values for polycyclic aromatic hydrocarbons. These SRM complex mixtures are available to scientists as reference standards for analytical chemistry research and are under consideration as SRMs for mutagenicity studies of complex environmental mixtures. This paper briefly describes the final study design, protocol, selection of the complex mixtures, and implementation of this international study.


Results of the IPCS collaborative study on complex mixtures. The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples--an air-particulate sample and a diesel-particulate sample--and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 17.5%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, +S9) to 6697 (TA100, +S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.


Genotoxicity of polycyclic aromatic hydrocarbons in Escherichia coli PQ37. In the present investigation, 32 polycyclic aromatic hydrocarbons (PAHs) were tested for genotoxicity in E. coli PQ37 using the standard tube assay of the SOS chromotest. PAHs such as benzo[ghi]fluoranthene, benzo[j]fluoranthene, benzo[a]pyrene, chrysene, dibenzo[a,l]pyrene, fluoranthene and triphenylene exhibited high genotoxicity when incubated in the presence of an exogenous metabolic activation mixture. The results were compared to those obtained with the Salmonella/microsome test.


Sources of variation in the mutagenic potency of complex chemical mixtures based on the Salmonella/microsome assay. Twenty laboratories worldwide participated in a collaborative trial sponsored by the International Programme on Chemical Safety on the mutagenicity of complex mixtures as expressed in the Salmonella/microsome assay. The U.S. National Institute of Standards and Technology provided homogeneous reference samples of urban air and diesel particles and a coal tar solution to each participating laboratory, along with samples of benzo[a]pyrene and 1-nitropyrene which served as positive controls. Mutagenic potency was characterized by the slope of the initial linear component of the dose-response curve. Analysis of variance revealed significant interlaboratory variation in mutagenic potency, which accounted for 57-96% of the total variance on a logarithmic scale, depending on the sample, strain and activation conditions. Variation among replicate extractions of organic material (required for the air and diesel particles) and among replicate bioassays within the same laboratory was also appreciable. The average potencies for air and diesel particles in laboratories using Soxhlet extracts were not significantly different from those in laboratories using sonication, although there was larger interlaboratory variation for the Soxhlet method. Repeatability (which approximates the coefficient of variation within laboratories) ranged from 18 to 40% for air and diesel particles extracted using sonication, depending on the strain and activation conditions. Repeatability of Soxhlet-extracted air and diesel particles, however, ranged from about 37 to 89% including outliers and from about 11 to 31% excluding outliers. Repeatability of the coal tar sample and the 2 positive controls was in the range 18-34%. Reproducibility (which approximates the coefficient of variation between laboratories) was generally at least twice repeatability, and exceeded 100% for Soxhlet-extracted air and diesel particles, as well as 1-nitropyrene. Reanalysis of the data omitting observations of more than 1500 revertants/plate generally had little effect on these results. Elimination of outlying observations had limited impact, with the exception of Soxhlet-extracted air and diesel particles. In this case, reproducibility of bioassay results was notably improved, due largely to the omission of results for replicate extractions which varied more than 5-fold within one laboratory. Normalization of the log potency slopes for the mixtures by the corresponding slopes for benzo[a]pyrene tended to reduce this variation, although variation was increased after normalization by 1-nitropyrene. Adjustment for the percentage of organic matter extracted from the air and diesel particulate samples had little effect on variation for sonication-extracted particles, whereas variation was reduced for diesel particles and increased for air particles for Soxhlet.


Genotoxicity of 1,3- and 1,6-dinitropyrene: induction of micronuclei in a panel of mammalian test cell lines. 1,3-Dinitropyrene (1,3-DNP) and 1,6-dinitropyrene (1,6-DNP) were assessed for their potential to increase the frequencies of micronuclei in a panel of test cell lines consisting of H4IIEC3/G-, 5L, 5L/r-1,3-DNP1, 208F, V79, V79/r-1,6-DNP1, HepG2 and BWI-J cells, which have been partially characterized for their expression of xenobiotic metabolising enzymes. The micronuclei were analyzed for the presence or absence of kinetochores indicating the occurrence of aneuploidy or chromosome breakage, respectively. 1,3-DNP caused a substantial increase in the frequency of micronuclei only in V79 cells. 1,6-DNP was strongly genotoxic in lines H4IIEC3/G-, 208F, V79 and, to a minor degree, in 5L/r-1,3-DNP1. It caused the formation of kinetochore-positive as well as kinetochore-negative micronuclei in V79 cells but only of kinetochore-negative micronuclei in H4IIEC3/G- and 208F cells. 1,6-DNP-induced formation of micronuclei was paralleled by the appearance of multinucleated cells. Treatment of V79 cells with 1,3-DNP resulted in the same types of damage as treatment with 1,6-DNP, although considerably higher concentrations were required. The results show that 1,6-DNP can be highly genotoxic in mammalian cells, whereas, at least in the panel of test cell lines used, 1,3-DNP possesses only a low genotoxic activity. 1,3-DNP appears to be activated to genotoxic products in V79 cells by the same pathway(s) as 1,6-DNP.


Overview, conclusions, and recommendations of the IPCS collaborative study on complex mixtures. The International Programme on Chemical Safety (IPCS) sponsored an international collaborative study to examine the variability associated with the extraction and bioassay of standard reference materials (SRMs) that are complex environmental mixtures provided by the U.S. National Institute of Standards and Technology (NIST). The study was also intended to evaluate the feasibility of establishing bioassay reference values and ranges for the SRMs. Twenty laboratories from North America, Europe, and Japan participated in the study. As part of the mandatory core protocol, each laboratory extracted the organic material from two particulate samples and bioassayed these extracts. A coal tar polycyclic aromatic hydrocarbon (PAH) solution and two mutagenic control compounds were also subjected to bioassay without prior extraction by the participating laboratories. The bioassay used was the Salmonella/microsomal plate incorporation assay. For the optional portion of the study, a laboratory was free to use the SRMs for any type of exploratory research. The primary purpose of the required portion of the study was to estimate the intra- and inter-laboratory variability in mutagenic potencies of the test materials and to determine whether or not the NIST mixtures could be used as reference materials by others performing the Salmonella assay. Repeatability (intra-laboratory variance) of the bioassay results ranged from 16% to 88% depending on the SRM and the bioassay conditions (tester strain and metabolic activation), whereas reproducibility (inter-laboratory variance) ranged from 33% to 152%. Between-laboratory variability was the main source of variation accounting for approximately 55-95% of the total variation for the three environmental samples. Variation in the mutagenic potency of the control compounds was comparable, with the exception of 1-nitropyrene for which the reproducibility ranged from 127% to 132%. In summary, NIST SRMs provided useful materials for an international inter-laboratory study of complex mixtures. By establishing both intra- and inter-laboratory variance for the mutagenicity results for these materials, the usefulness of these SRMs as reference materials for the Salmonella bioassay was established, critical procedures within the bioassay protocol were identified, and recommendations for future efforts were delineated.


Characterization of organic extracts from standard reference materials 1649, 'urban dust/organics,' and 1650, 'diesel particulate matter', using a microsuspension assay. A WHO/IPCS/CSCM study. Mutagenicity associated with replicate organic extracts from standard reference materials 1649 'urban dust/organics' (air particles), and 1650, 'diesel particulate matter' (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation assay. In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98-S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/micrograms, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/microgram, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 -S9 but the TA98-1,8-DNP6 levels were.


Influence of different occupations with possible mutagenic effects on reproduction and level of induced chromosomal aberrations in peripheral blood. In a group of 200 dysfertile couples (400 persons), the possible role of different occupations in failures of reproduction was assessed. These couples were examined from different points of view, classical genetic examination (pedigree, kayrotype, etc.) included. The suspected genotoxic effects in the personal history were checked also by testing the level of induced chromosomal aberrations. A significantly increased level of induced chromosomal aberrations was detected in 37 persons, i.e., 9.3% of the whole group under study. The average level of induced aberration in these subjects was 6.8%, as opposed to the control group (fertile and dysfertile persons without any unusual exposure to mutagens) with a mean of 1.58% aberrant cells in peripheral blood. Most of the occupations with demonstrated genotoxic effects involve daily contact with chemicals of different types. In some persons also intensive therapy in the recent past had genotoxic effects.