Changes in cortical activity when subjects scan memory for tones. The magnetoencephalogram (MEG) was used to detect regional changes in spontaneous cortical activity accompanying short-term memory search. This method was chosen because magnetic fields are detectable only within a few centimeters of the projections of their sources onto the scalp. The specific hypothesis that auditory cortex is involved in scanning memory for tones was tested by sensing the field of the magnetic counterpart to N100 (N100m) which is known to originate in auditory cortex. N100m was measured at many different positions and the spontaneous cortical rhythms in the alpha bandwidth (8-12 Hz) were measured at the same places. These rhythms were found to be suppressed while subjects scanned memory for musical tones in a Sternberg paradigm. For 3 subjects, both the MEG suppression time (ST) and reaction time (RT) increased linearly with memory set size. The correlation between ST measured over the left hemisphere and set size was significant for two subjects but not significant for the third, and the slopes of the regression lines relating ST to set size were too shallow to be related to the time required to scan memory. However, the correlation between ST of the right hemisphere and set size was highly significant for all subjects, and the slopes of the regression lines were comparable to those relating RT to set size. The electroencephalogram (EEG) recorded with midline electrodes failed to reveal a significant relationship between suppression time and set size for 2 of the subjects, thus ruling out global alpha blockage and generalized arousal as the basis for the task-related suppression duration. The electric N100, measured at Cz, decreased significantly in amplitude with set size for 2 subjects, but it increased significantly in amplitude for the third subject. In contrast, RT increased with set size for all subjects. N100m measured over the right hemisphere was similar to the behavior of N100, while N100m measured over the left hemisphere showed little change in amplitude with set size, thus establishing an asymmetry in N100 between the hemispheres. Since N100 amplitude is normally larger when attention is paid to auditory stimuli, differential attention alone cannot account for the relation between ST and set size. Furthermore, the processing negativity, which may be superimposed on N100 in selective attention tasks, was not discernible for any set size. It was also found that ST prior to the button press was not correlated with RT. Hence, the covariation of set size with ST is not attributable to preparation for a motor response.(ABSTRACT TRUNCATED AT 400 WORDS)


Antibodies against terminal galactosyl (alpha 1-3) galactose epitopes in systemic sclerosis (scleroderma). Sera from 224 patients with systemic sclerosis (scleroderma) were analyzed for circulating antibodies against an antigenic determinant characterized by two molecules of galactose in alpha 1-3 linkage. About 45% of the patients were found to have values above the normal range. The mean antibody level was significantly higher than that found in normal subjects (p less than 0.001) or in patients with primary Raynaud's phenomenon who were included as controls. The mean level of anti-Gal antibodies correlated with the degree of skin and internal organ involvement, as well as with the presence of progression or inflammation. Furthermore, when patients with early onset disease were analyzed, high levels of anti-Gal antibodies were present in the subgroups characterized by evidence of progression or inflammation, whereas patients with stable disease did not differ from the controls. We conclude that humoral immunity against Gal alpha 1-3 Gal is an early feature of scleroderma, may be important for its pathogenesis, and may provide a more sensitive tool to detect disease activity.


Chlamydial rRNA in the joints of patients with Chlamydia-induced arthritis and undifferentiated arthritis. Synovial fluid and synovial membrane specimens of 11 patients with Chlamydia-induced arthritis (CIA), 24 patients with undifferentiated arthritis (UndA), 4 patients with post-enteritic reactive arthritis, 3 patients with Lyme arthritis and 9 patients with rheumatoid arthritis were investigated for the presence of Chlamydia trachomatis (C. trachomatis). A single stranded DNA-probe was used for nucleic acid hybridization with ribosomal RNA (rRNA) from C. trachomatis. In 4 patients (CIA = 1, UndA = 3) chlamydial rRNA was found in the synovial fluid. In one additional patient (CIA) the specimen of a synovial membrane biopsy was positive for chlamydial rRNA. The detection of intra-articular chlamydial rRNA is discussed as an indicator for the presence of viable Chlamydiae in inflamed joints.


Cortical DC potential shifts accompanying auditory and visual short-term memory. Negative DC potential shifts appeared over the scalp during the performance of verbal and non-verbal short-term memory tasks. Three items were successively presented (presentation of memory items) and then had to be retained in memory for 3 sec (memory retention) before being compared to a probe which was either a member (in set) or not a member (out of set) of the memory set. Verbal items (the digits "1" through "9") were tested in the auditory and visual modality and non-verbal items (musical notes) were tested in the auditory modality. Stimulus modality had a significant effect on DC potential shifts during both presentation of memory items and memory retention. There was a sustained negative shift during these periods which was larger over frontal regions with auditory than with visual material whereas the negative shift was larger over posterior temporal regions with visual than with auditory material. Out of 21 subjects who participated in the study, 9 reported the use of visual images in the auditory task, 5 used subvocal auditory rehearsal in the visual task and 7 used imagery concordant with the stimulus modality being memorized. These different strategies had a significant effect on the amplitudes and distribution of the DC potential shifts. The speed of response affected the amplitude of the DC potential shifts in the frontal regions, being larger with fast RTs than with slow RTs but only when verbal items were being processed. These results indicate that stimulus modality, modality of mental imagery, and speed of scanning of the memory store affect DC potential shifts during a 3 sec period of memory retention.


Properties of neuronal activity in cortex and subcortical nuclei of the human brain during single-word processing. Neuronal impulse activity (NIA) in different cortical areas, subcortical nuclei of the thalamus and striopallidum was recorded via depth electrodes in human patients as they performed a visual word recognition task. The properties of neuronal responsiveness were compared across regions. During single-word processing approximately 75-80% of the responsive cortical neurons were characterized by poststimulus inhibition of firing rate; an increased firing rate (i.e., excitatory response) was observed in about 20-25% of the responsive cortical neurons. Among the subcortical nuclei, mainly in the thalamus and striopallidum, more than 90% of the responsive neurons were characterized by poststimulus excitation; less than 10% exhibited poststimulus inhibition of firing rate.


Event-related desynchronization: the effects of energetic and computational demands. The effects of a subject's activation state on cognitive processing were studied, while subjects performed verbal and non-verbal tasks under a speed and accuracy instruction. It was found that stressing speed influenced the level of prestimulus alpha power and consequently the amount of relative event-related desynchronization (ERD). Increasing task complexity led to an increase in the amount and duration of relative ERD. Both prestimulus level of alpha power and relative ERD were asymmetrically distributed over the left and right hemispheres. No verbal/non-verbal task-dependent asymmetries in phasic ERD were found. The data suggest that the level of prestimulus alpha power is mainly influenced by the subject's activation state, whereas relative ERD mainly reflects phasic changes in cognitive processing.


Unilateral 14 and 6 Hz positive bursts.

Unilateral 14 and 6 Hz positive bursts. We describe the unilateral occurrence of 14 and 6 Hz positive bursts in successive EEGs in a 25-year-old woman following surgical resection of a left parietal arteriovenous malformation which had caused a left parieto-temporal intracerebral hematoma. This is only the second reported case of unilateral 14 and 6 Hz positive bursts. This could represent either a normal pattern seen unilaterally because of a skull defect or be a manifestation of neuronal damage.


Prediction of the side of hand movements from single-trial multi-channel EEG data using neural networks. Thirty channels of EEG data were recorded prior to voluntary right or left hand movements. Event-related desynchronization (ERD) was quantified in the 8-10 Hz and 10-12 Hz bands in single-trial data and used as training input for a neural network comprised of a learning vector quantizer (LVQ). After a training period, the network was able to predict the side of hand movement from single-trial EEG data recorded prior to movement onset.


Selective effect of poly(lysine) on the enhancement of the lyn tyrosine protein kinase activity. Increased specificity toward src peptides. A tyrosine protein kinase (TPK-I), isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and recently identified as the product of the lyn oncogene [Brunati, A. M., Donella-Deana, A., Ralph, S., Marchiori, F., Borin, G., Fischer, S. & Pinna, L. A. (1991) Biochim. Biophys. Acta 1901, 123-126], is stimulated by a variety of effectors, including poly(lysine), heparin and very high NaCl concentrations. The efficacy of these compounds is variably dependent on the nature of the phosphoacceptor peptide substrates. Here we show that poly(lysine), but neither NaCl nor heparin, specifically enhances the phosphorylation efficiency of lyn TPK for the peptide EDNEYTA (src peptide). It reproduces the main autophosphorylation site of pp60c-src (Tyr416), which is entirely conserved in lyn TPK. The favourable effect of poly(lysine) is accounted for by both a dramatic drop of the Km value (70 microM versus 670 microM) and more than a threefold increase in Vmax. The effect is not so evident with a variety of different peptides, either unrelated to the src peptide (e.g. angiotensin II, AAYAA) or derived from the src peptide by single or multiple substitutions of the residues located on the N-terminal side of tyrosine. In particular, the responsiveness to poly(lysine) is dramatically reduced whenever alanine is replaced for either asparagine at position -2 or glutamic acid at position -1, either in the src heptapeptide or in its shorter derivative, the pentapeptide NEYTA. In sharp contrast, the favourable effect of 2 M NaCl, which is invariably accounted for only by an increased Vmax, is especially evident with peptides like angiotensin II and AAYAA, whose phosphorylation is less sensitive to poly(lysine) stimulation. The phosphorylation of the src peptides are actually inhibited rather than stimulated by 2 M NaCl. Consistent with this, lyn TPK autophosphorylation is also dramatically stimulated by poly(lysine) while being inhibited by 2 M NaCl. These data show that poly(lysine) deeply alters the selectivity of lyn TPK by conferring to it an enhanced activity and an especially high affinity toward peptides that reproduce the conserved autophosphorylation site of the TPK of the src family. It is suggested that endogenous compound, whose effect is mimicked by poly(lysine) in vitro, may play a relevant role in determining the specificity of lyn TPK in vivo and possibly of other TPK of the src family.


Tissue distribution and change in potato starch phosphorylase mRNA levels in wounded tissue and sprouting tubers. Starch phosphorylase has been cloned from a lambda gt10 cDNA library of potato tuber mRNA. Selected recombinants have been used to demonstrate that phosphorylase mRNA is most abundant in tubers but is also detectable in stolon, root, stem and leaf tissue. The level of phosphorylase mRNA was greatly reduced in wounded stem and tuber tissue. The wounding-induced decrease in phosphorylase mRNA levels is not reversed in the presence of sucrose or mannitol. Regional differences are described in the levels of phosphorylase and patatin mRNA in different parts of the tuber and in the shoot of sprouting potatoes.


Interleukin-7 expression during mouse thymus development. We have monitored the expression of interleukin-7 (IL-7) in the developing embryonic mouse thymus by a combination of quantitative polymerase chain reaction (PCR) and immunofluorescence microscopy. A strong specific signal for IL-7 mRNA was detected by day 12 in the developing fetal thymus. IL-7 mRNA was found to be maximally expressed on day 15, and then decreased over the next 5 days. Immunofluorescence staining of fetal thymus sections using an anti-IL-7 antibody confirmed these PCR data. IL-7 protein expression was first detected at day 13 of development. At 14 days the intensity of the staining increased by a factor of three and stayed at this level over the next 4 days. The same anti-IL-7 antibody used for immunofluorescence, blocked the proliferation of fetal thymocytes in organotypic cultures. In addition, we detected mRNA coding for IL-2 and SCF (also known as the steel factor or KL) in embryonic thymocytes. The implications of these findings for early thymocyte growth are discussed.


Antibody binding to a collagen type-II epitope gives rise to an inhibitory peptide for autoreactive T cells. It is well documented that antigen recognition by T cells requires small peptides which are generated by protein cleavage in antigen-presenting cells. These peptides have to associate with major histocompatibility complex (MHC) molecules in order to be recognized. An inhibitory peptide may bind to the same site of the MHC-encoded protein but is not recognized by the T cell. Here we describe a stimulatory and an inhibitory peptide sequence within human collagen type II (CII) as defined by means of the same autoreactive human T cell clone. Most interestingly, the inhibitory peptide is not generated by regular processing in peripheral blood mononuclear cells but only in the presence of an antibody that binds to the same domain and thereby seems to protect the inhibitory sequence. This finding may indicate that certain autoantibodies have the potential to block autoreactive T cells with specificity for a distinct epitope on the same antigen.


Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis. Myelin basic protein (MBP)-autoreactive T cells have a crucial pathogenetic role in experimental allergic encephalomyelitis (EAE) and certain MBP epitopes may be immunodominantly recognized. The heterogeneity and quantity of the T cell response to different epitopes of MBP in multiple sclerosis (MS) and non-MS controls is not so clearly defined. We now study T cell reactivity to six different peptides of MBP in MS compared to controls in short-term cultures of blood mononuclear cells by measuring numbers of T cells that secrete interferon-gamma in response to antigen. In comparison with controls, MS patients showed dramatically increased numbers of MBP peptide-reactive T cells with mean values varying between 10.4 and 22.5 per 10(5) blood mononuclear cells. Among those MBP peptides examined (amino acid 1-20, 63-88, 89-101, 96-118, 110-128 and 148-165), no single peptide is preferentially recognized. Neither is any preferential response apparent after subdivision of the MS patients according to their HLA-DR genotype. Our findings suggest that a quantitative increase of a broad repertoire of myelin-autoreactive T cells with capacity to secrete IFN-gamma can be important for the pathogenesis of MS.


Tissue-specific migration pathways by phenotypically distinct subpopulations of memory T cells. A proportion of T cells recirculate in a tissue-selective manner. Recent studies which showed that the skin-tropic subset of T cells was of memory/activated type, led us to examine whether the preferential homing of T cells to the gut also involved memory T cells, and if so whether these memory T cells were phenotypically distinct from other memory T cells. Lymphocytes migrating through the gut and the skin of sheep was collected by cannulating the lymphatic ducts draining these tissues. Both naive and memory T cells were found to recirculate through the gut, although only memory T cells migrated through the skin. However, when T cells from the gut were labeled with fluorescein isothiocyanate and assessed for their migration back to the gut, it was the memory population which showed a tropism for the gut. Gut-tropic memory T cells migrated poorly through the skin, indicating that these cells were distinct from skin-tropic memory T cells. This was confirmed by phenotypic analysis. Gut memory T cells expressed very low levels of the alpha 6 and beta 1 integrins, in contrast to skin memory T cells which expressed high levels. There was no evidence for heterogeneity within the naive T cell population, which migrated preferentially to lymph nodes. This migration pattern could be explained in part by the high expression of the L-selectin (lymph node homing receptor, LAM-1) on naive T cells, in contrast to memory T cells from gut or skin which were mostly L-selectin negative. These results in sheep indicate that subsets of alpha/beta memory T cells show tissue-selective migration patterns, which probably develop in a particular environment following encounter with antigen.


Identification of the nonamer peptide from influenza A matrix protein and the role of pockets of HLA-A2 in its recognition by cytotoxic T lymphocytes. Influenza matrix peptide 58-66 is shown to be the optimal nonamer for binding to HLA-A2 and presentation to cytotoxic T lymphocytes (CTL). If titered out to 2 x 10(-10) - 4 x 10(-10) M in CTL-mediated lysis assays and to 3 x 10(-9) M in an HLA-A2 assembly-stabilization assay in cell lysates. The peptide was shown to make probable contacts with its carboxy terminus close to residue 116 in the floor of the cleft of HLA-A2, close to the F pocket. The side chain of the amino-terminal amino acid was unimportant, but its free amino and carbonyl groups in the A pocket appeared important in optimizing peptide presentation. The B pocket probably accommodates the side chain of residue 2 (isoleucine) and was shown to be critical in peptide presentation.


T cell epitope selection: dominance may be determined by both affinity for major histocompatibility complex and stoichiometry of epitope. The majority of T cell hybridomas produced in the BALB/c mouse in response to immunization with lambda repressor cI recognize a peptide fragment comprising of residues 12 to 26 (P12-26). Some other parts of the cI (P1-14, P33-48 and P73-88) are defective in generating T cell responses in the BALB/c mouse. P73-88 may be converted into a T cell determinant if a few more amino acid residues are included (P67-88). Together with P46-67 and P80-102, most peptides derived from cI were capable of eliciting T cell responses by themselves in BALB/c mouse. The mechanisms underlying the selection of P12-26 over the other epitopes when lambda repressor was used as immunogen were examined. The dominant response to P12-26 was attenuated by tolerizing with intravenous administration of P12-26. Under such treatment the T cell response to P12-26 was reduced by 80% but there was no enhancement on the responses toward other epitopes. The selection of P12-26 is, thus, unlikely to be due to a competition at the T cell level. It was also found that the dominance of P12-26 was not simply due to a higher affinity of P12-26 for major histocompatibility complex molecules. For example P12-26 binds better to I-Ad molecule than P80-102, but co-injection with equimole of P12-26 only slightly inhibited P80-102-induced T cell response. Instead, it required a few molar excess of P12-26 to effectively block the association of P80-102 with I-Ad molecules and to inhibit the T cell immunity to P80-102. Since epitopes such as P46-67, P67-88 and P80-102 were generated from lambda repressor cI at a lower molar basis than that of P12-26, it is suggested that the dominance of P12-26 was probably generated by such stoichiometry difference, in addition to the higher affinity of P12-26 for I-Ad molecules.


Interferon-gamma regulates expression of a novel keratin class I gene. Interferon (IFN)-gamma has been implicated in the pathogenesis of several autoimmune disorders and inflammatory skin diseases. To identify novel mediators involved in the IFN-gamma response we have used differential hybridization of a cDNA library prepared from IFN-gamma-treated HeLa cells to isolate a gene that is induced following treatment with IFN-gamma. We report here the molecular cloning and characterization of a cDNA detecting a 1.6-kb mRNA that accumulated in response to IFN-gamma but not in response to IFN-alpha or IFN-beta. The gene is regulated by IFN-gamma in human cell lines of epithelial origin. The mRNA encodes a predicted protein of 432 amino acids and the primary structure of the protein demonstrates that it is a novel member of developmentally regulated keratin class I genes.


Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-beta. RAW 264.7 macrophages induced with lipopolysaccharide and interferon-gamma expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine. Transforming growth factor-beta 1 prevented the induction of both enzymes.


Expression analysis of ruminant alpha-lactalbumin in transgenic mice: developmental regulation and general location of important cis-regulatory elements. The bovine alpha-lactalbumin transgene with 750 bp and 336 bp of the 5' and 3' flanking region, respectively, is developmentally regulated as its endogenous counterpart in transgenic mice. Comparative expression analysis of three 5'-shortened constructs suggests that the region -477/-220 contains important cis-acting transcriptional elements. The level of expression of a long caprine alpha-lactalbumin transgene encompassing 8.5 kb and 9.5 kb of the 5' and 3' flanking region, respectively, was higher but still unrelated to the copy number. Expression of the transgenes and of endogenous milk-protein genes was tissue-specific. In contrast with a recent report, only low amounts of the relevant mRNA were detected in some skin samples, which suggests a possible contamination by mammary tissue.


Induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in vascular smooth muscle cells. Inducible vascular nitric oxide synthase accounts for the contractile impairment observed in endotoxemia. We provide evidence that lipoteichoic acid (LTA) from Staphylococcus aureus, a micro-organism without endotoxin, also induces nitric oxide synthase. Our study demonstrates that on endothelium-free rings of rat aorta. LTA-like lipopolysaccharide induces a loss of contractility restored by Methylene blue and NG-nitro-L-arginine-methyl ester (LNAME). Moreover in cultured vascular smooth muscle cells, LTA produces a dose-dependent increase in intracellular cyclic GMP which is antagonized by LNAME and prevented by dexamethasone.


Effect of ploidy on transcription levels in cultured rat aortic smooth muscle cells. Aging and hypertension increase the number of polyploid smooth muscle cells (SMC) in a blood vessel. We assessed the effect of ploidy on the transcription of several genes in SMC cultures derived from newborn and adult rats. In diploid and tetraploid subcultures of SMC from newborn rats, RNA expression of the genes assayed is linked with ploidy. However, when phenotypically different SMC cultures derived from newborn and adult rats were compared, transcription levels varied from gene to gene and not linked with the ploidy. Thus, differences in gene expression due to polyploidy are superimposed on those due to other phenotypical features.


A novel 15N-labeling method to selectively observe 15NH2 resonances of proteins in 1H-detected heteronuclear correlation spectroscopy. An experimental method to selectively label side-chain NH2 groups of glutamine and asparagine in proteins with 15N is proposed. This selective labeling method enables to observe only 15NH2 resonances and thus, to discriminate between 15NH and 15NH2 resonances in a 1H-detected heteronuclear correlation spectrum. This method gives results with approximately two times higher sensitivity than those obtained by elaborate pulse sequences such as DEPT-HSQC and will be useful for studying the molecular interaction involving the side chains of Asn and Gln residues.


Alternative promotion of aromatase P-450 expression in the human placenta. We have previously reported the isolation and characterization of the human gene encoding aromatase cytochrome P-450 (P-450AROM). The gene had been demonstrated to span at least 52 kb and contain ten exons, the first of which, exon I.1, is untranslated. Here we report the isolation and characterization of a P-450AROM cDNA from a human placental primer-extended cDNA library which contains a unique 5' sequence. This cDNA has been isolated and sequences used to screen a human placental genomic library for the presence of a unique first exon. The exon (exon I.2) lies 9 kb 5' of the second, ATG-containing exon (exon II) and is spliced onto exon II at the same site as that reported for exon I.1. DNA sequence analysis indicates that exon I.2 has a putative TATA (TAAA) sequence 33 base pairs (bp) upstream from a putative transcription start site and putative CAAT (CATT) binding sequence beginning at 54 bp upstream from this start site. Polymerase chain reaction (PCR) amplification experiments indicate that mRNA containing exon I.2-specific sequences can be demonstrated in tissues of fetal, but not adult, origin. These data have been confirmed by Northern analysis in the placenta. Characterization of this genomic clone containing exons I.2 and II now establishes the P-450AROM gene to be at least 72 kb in length and raises new questions regarding tissue specific and developmental control of aromatase expression in the human.


The role of the basement membrane in differential expression of keratin proteins in epithelial cells. Extracellular matrix is considered to play an important role in determining the phenotype of cells with which it interacts. Here we have investigated the possibility that extracellular matrix is involved in specifying the pattern of keratin expression in epithelial cells. For these studies, we have developed an explant system in which epithelial cells from one type of stratified epithelial tissue, namely conjunctiva, are maintained on an extracellular matrix substrate derived from a different tissue, namely cornea. These ocular tissues are ideal for such analyses since they express distinct sets of keratins. For example, bovine conjunctival epithelium processed for immunofluorescence is not recognized by antibody preparations against keratin K3 or K12. In contrast, K3 and K12 antibodies generate intense staining in bovine corneal epithelium. At the immunochemical level, conjunctival cells in situ appear to possess no K12 and only trace amounts of K3, whereas corneal epithelial cells in situ possess both K3 and K12. When conjunctival cells are maintained on a corneal substrate with an intact basement membrane for 10 days in vitro they begin to express keratin K12 as determined by immunofluorescence. On the other hand, conjunctival cells that are maintained on a corneal substrate lacking a basement membrane fail to show staining with K12 antibodies. Conjunctival cells begin to show intense staining using K3 antibodies within about 10 days of being placed in culture regardless of their substrate. These results indicate that basement membrane can play a positive role in determining cell-specific expression of certain keratins such as K12. However, other keratins such as K3 may be "unmasked" and/or their expression may be upregulated simply by placing conjunctival epithelial cells in culture. We speculate that in conjunctiva K3 expression is influenced by certain negative exogenous factors. We discuss the possible means of regulation of keratin expression in our model system.


Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons. We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.


An ecdysteroid-inducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily. Using cDNAs for the human retinoic acid receptor alpha (hRAR alpha) and Drosophila hormone receptor 3 (DHR3), we isolated a cDNA encoding a member of the steroid hormone receptor superfamily from the tobacco hornworm, Manduca sexta. Sequencing showed that this cDNA is most closely related to DHR3 (97 and 68% amino acid identity in the DNA and ligand binding regions, respectively) followed by hRAR alpha (65 and 20% identity, respectively) and therefore is named MHR3. The cDNA hybridized to two mRNAs (3.8 and 4.5 kb) found in the epidermis during the ecdysteroid rises for the embryonic, larval, and pupal molts. Culture of fourth instar larval epidermis with 4 microM 20-hydroxyecdysone (2 micrograms/ml 20HE) caused the appearance of MHR3 mRNA within 3 hr and maximal expression by 6 hr; after 12 hr continuous exposure to 20HE, the mRNA level declined. The 4.5-kb mRNA appeared first, both were present in equal amounts by 12 hr, and by 20 hr the predominant transcript was 3.8 kb. Similar 20HE-induced expression was seen in epidermis explanted 1 day after the onset of wandering, although with a slower time course. The induction was largely independent of protein synthesis, but the subsequent decline required protein synthesis as is typical of the "early" puffs in Drosophila. Continuous exposure to 20HE was necessary for MHR3 expression; in its absence, the mRNA declined with a half-life of 2 hr. Thus, MHR3 is an ecdysteroid-inducible DNA binding protein that likely is a transcription factor involved in the cascade of gene activation and inactivation caused by ecdysteroids during the insect molt.


Induction of avascular yolk sac due to reduction of basic fibroblast growth factor by retinoic acid in mice. Vasculogenesis depends on autocrine secretion of basic fibroblast growth factor (bFGF) from capillary endothelial cells. Retinoic acid (RA) induced avascular yolk sac (AVY) of mouse embryos of dams given 60 mg/kg of RA orally on Day 8 of gestation and sacrificed 3 days later. We studied the localization and transcriptional expression of bFGF and FGF-receptor (flg), heparin-binding growth factor (HBGF) activity, localization of lysosomal enzymes and alpha 1-antitrypsin (AAT), and electron microscopy of the normal mouse visceral yolk sac (VYS) and AVY. bFGF, which is normally present in the endoderm of the VYS of 8-day-old embryos and in all components of the VYS by Day 11 of gestation, was reduced in the AVY. However, in the presence of bFGF in vitro capillary nets were restored in the AVY. The mRNA for bFGF was not detectable in either VYS or AVY, while flg mRNA was detected equally in both organs in Northern blotting. The characteristic distribution pattern of lysosomal enzymes, acid phosphatase, lysozyme, and cathepsin D, and AAT was altered in the AVY. The level of acid phosphatase and AAT was reduced to 10% in the AVY. Electron microscopy revealed a partial or total loss of lysosomal membranes where the contents of lysosomes fused with adjacent lysosomes and the external organelles. These results suggest that vitelline blood vessels are not developed by endogenous autocrine bFGF but by exogenous transcellular bFGF from absorptive endodermal cells. Retinoic acid does not affect the angiogenic capacity of the VYS mesenchyme but destroys lysosomes, which release hydrolytic enzymes, leading to degradation of AAT in the endodermal cells and then digestion of endocytosed bFGF.


Glucose and insulin chronically regulate insulin action via different mechanisms in BC3H1 myocytes. Effects on glucose transporter gene expression. We previously reported that, in primary cultured adipocytes, chronic exposure to glucose plus insulin impairs the insulin-responsive glucose transport system. In this study, we examined regulation of glucose transport in BC3H1 myocytes as a model for muscle and found important differences between BC3H1 cells and adipocytes. In myocytes, chronic glucose exposure per se (25 mM) decreased basal glucose transport activity by 78% and insulin's acute ability to maximally stimulate transport by 68% (ED50 approximately 2.5 mM; T1/2 approximately 4 h). D-Mannose and 3-O-methyl-glucose diminished transport rates with approximately 100 and 50% of the potency of D-glucose, respectively, whereas L-glucose, D-fructose, and D-galactose were inactive. Chronic glucose exposure also reduced cell surface insulin binding by 30% via an apparent decrease in receptor affinity, and this effect was associated with a comparable rightward shift in the insulin-glucose transport dose-response curve. In other studies, persistent stimulation with 15 nM insulin also decreased maximally stimulated glucose transport activity, which was independent and additive to the regulatory effect of glucose. Moreover, glucose and insulin-induced insulin resistance via different mechanisms. Glucose (25 mM) reduced the number of cellular glucose transporter proteins by 84% and levels of GLUT1 transporter mRNA by 50% (whether normalized to total RNA or CHO-B mRNA). In contrast, chronic insulin exposure led to a 2.1-fold increase in GLUT1 mRNA but did not alter cellular levels of transporter protein. Cotreatment with glucose prevented the insulin-induced rise in GLUT1 mRNA. BC3H1 cells did not express GLUT4 mRNA that encodes the major transporter isoform in skeletal muscle. In conclusion, in BC3H1 myocytes 1) glucose diminished insulin sensitivity by decreasing insulin receptor binding affinity and decreased basal and maximally insulin-stimulated glucose transport rates via cellular depletion of glucose transporters and suppression of GLUT1 mRNA; 2) chronic insulin exposure exerted an independent and additive effect to reduce maximal transport activity; however, insulin increased levels of GLUT1 mRNA and did not alter the cellular content of glucose transporters; and 3) although BC3H1 cells are commonly used as a model for skeletal muscle, studies examining glucose transport should be interpreted cautiously due to the absence of GLUT4 expression. Nevertheless, the data generally support the idea that, in non-insulin-dependent diabetes mellitus, hyperglycemia and hyperinsulinemia can induce or exacerbate insulin resistance in target tissues.


Multiple TCR V beta usage by infiltrates of young NOD mouse islets of Langerhans. A polymerase chain reaction analysis. Because a restricted repertoire of T-cell receptor (TCR) V beta gene expression has been reported in other autoimmune diseases, the possibility of similarly restricted V beta gene expression by T-cell infiltrates of NOD mouse islets was examined. With isolated islets from 4- to 12-wk-old NOD mice, a prospective polymerase chain reaction analysis with 18 V beta-specific oligonucleotide primers was performed on the noncloned and unexpanded islet-infiltrating T cells. The methodology used permitted the detection of a minimum of 50 T cells. In contrast to the restricted TCR V beta gene usage reported for other autoimmune diseases, infiltrates of even the youngest mice were characterized by expression of multiple V beta gene segments.


16,16-Dimethyl prostaglandin E2 reduces bile acid-mediated intestinal vascular injury in rats. To examine the effects of prostaglandin on bile acid-mediated intestinal vascular injury, male rats were given 50 mg/kg of fluorescein isothiocyanate (FITC)-stained dextran 70 or 25 mg/kg of Evans Blue intravenously. Before intestinal injury with 45-minute perfusion of 5 mmol/L chenodeoxycholic acid, rats received 16,16-dimethyl prostaglandin E2 (5 micrograms/kg intravenously or 0.5 micrograms/mL or in the perfusate for 15 minutes or vehicle). FITC-dextran clearance from the blood to the intestinal lumen and tissue Evans Blue content were used as measures of intestinal vascular injury. Morphological mucosal injury was assessed by transmission electron microscopy and quantitative histological analysis. Chenodeoxycholic acid perfusion caused villous denudation and shortening of and ultrastructural damage to villous venules. Functional vascular injury was evidenced by a 10-fold increase in the rate of FITC-dextran blood-to-lumen clearance and a 3-4-fold increase in tissue Evans Blue content. Pretreatment with either intravenous or intraluminal 16,16-dimethyl prostaglandin E2 reduced FITC-dextran clearance by 70%-80% and tissue Evans Blue content by 50%. However, only luminal prostaglandin reduced superficial mucosal morphological injury, possibly because of differences in the local concentrations of 16,16-dimethyl prostaglandin E2 or chenodeoxycholic acid or because of superficial mucosal protection and injury being, at least in part, independent of mucosal microvascular injury and protection.


Cyclic interdigestive pancreatic exocrine secretion: is it mediated by neural or hormonal mechanisms? Cyclic interdigestive exocrine pancreatic secretion and duodenal motility are closely linked. However, the mechanisms controlling this association are not well understood. The aim of this study was to determine whether a neural or hormonal mechanism controls the temporal association of interdigestive secretion and duodenal motility. In five dogs, the pancreas was autotransplanted to the pelvis with anastomosis of the pancreatic duct orifice to the bladder. Electrodes were positioned to monitor motility patterns of the in situ duodenum. After 10 days, dogs were studied on four occasions during fasting. Pancreatic output of amylase activity continued to cycle, but the periodicity of enzyme peaks (mean +/- SE) was different from the period of the duodenal migrating motor complex (MMC) (60 +/- 3 vs. 125 +/- 7 minutes; P less than 0.05). When grouped according to phase of duodenal MMC, amylase output per 10 minutes during phase I was significantly less than the outputs during phase II or III (135 +/- 52, 214 +/- 78, and 228 +/- 73 x 10(3) U; P less than 0.05). However, there was no temporal relationship of the cyclic output of amylase to duodenal phase III. No differences were found when amylase output was analyzed for the 30 minutes before phase III compared with the 30 minutes after phase III (687 +/- 253 vs. 378 +/- 110 x 10(3) U; P greater than 0.05). Plasma motilin concentrations varied with duodenal MMC, but no relationship existed between plasma motilin or plasma pancreatic polypeptide and peaks in amylase output. This study suggests that the close temporal coordination of interdigestive pancreatic exocrine secretion and duodenal motility is controlled primarily by a neural mechanism.


Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui. The halophilic archaebacterium, Haloarcula marismortui, contains two nonadjacent ribosomal RNA operons, designated rrnA and rrnB, in its genome. The 16S rRNA genes within these operons are 1472 nucleotides in length and differ by nucleotide substitutions at 74 positions. The substitutions are not uniformly distributed but rather are localized within three domains of 16S rRNA; more than two-thirds of the differences occur within the domain bounded by nucleotides 508 and 823. This domain is known to be important for P site binding of aminoacylated tRNA and for 30-50S subunit association. Using S1 nuclease protection, it has been shown that the 16S rRNAs transcribed from both operons are equally represented in the functional 70S ribosome population. Comparison of these two H. marismortui sequences to the 16S gene sequences from related halophilic genera suggests that (i) in diverging genera, mutational differences in 16S gene sequences are not clustered but rather are more generally distributed throughout the length of the 16S sequence, and (ii) the rrnB sequence, particularly within the 508-823 domain, is more different from the out group sequences than is the rrnA sequence. Several possible explanations for the evolutionary origin and maintenance of this sequence heterogeneity within 16S rRNA of H. marismortui are discussed.


Molecular analysis of a Salmonella enterica group E1 rfb gene cluster: O antigen and the genetic basis of the major polymorphism. Salmonella enterica is highly polymorphic for the O antigen, a surface polysaccharide that is subject to intense selection by the host immune system. This polymorphism is used for serotyping Salmonella isolates. The genes encoding O antigen biosynthesis are located in the rfb gene cluster. We report here the cloning and sequence of the 19-kb rfb region from strain M32 (serovar anatum, group E1) and compare it with that of strain LT2 (serovar typhimurium, group B). Genes for biosynthetic pathways common to both strains are conserved and have very similar sequences. In contrast, the five genes for CDP-abequose synthesis, present in strain LT2, are absent in strain M32; three open reading frames (ORFs) of strain LT2, thought to include genes for transferases, are not present in strain M32 but are replaced by three different ORFs with little or low level of similarity. Both rfb gene clusters are low in G + C content, indicating that they were transferred from a common ancestral species with low G + C content to S. enterica relatively recently (in the evolutionary sense). We discuss the recombination and lateral transfer events which may have been involved in the evolution of the polymorphism.


Initiator oligonucleotides for the combination of chemical and enzymatic RNA synthesis. Transcription reactions with T7 RNA polymerase were performed in the presence of short oligonucleotides (oligos) with guanosine at the 3'-end. We obtained transcripts which had included these 'initiator oligos' at their 5'-termini. The oligos could contain mixtures of deoxyribo-, ribo-, 2'-O-methylated and biotinylated nucleotides. Only the 3'-terminal guanosine of these oligos was encoded in the template DNA at the transcription start point, in contrast to the remainder of the sequence. This 5'-terminal sequence is variable and eliminates the limitation that transcripts must start with a 5'-terminal guanosine. With a 5'-biotinylated dinucleotide, we obtained end-labeled RNAs suitable for nonradioactive RNA sequencing.


Vitronectin in liver disorders: biochemical and immunohistochemical studies. The concentration of plasma vitronectin was determined and compared with various parameters of liver function including the blood coagulation system in patients with liver diseases. The severity of cirrhosis was graded according to Child's criteria and compared with the plasma vitronectin level. Furthermore, the distribution of vitronectin in the liver of patients with liver diseases was studied by light and electron microscopy using the indirect immunoperoxidase method. The plasma vitronectin level was low in all liver disease groups as compared with the healthy controls. The difference from the controls was significant in patients with hepatocellular carcinoma and decompensated cirrhosis. Moreover, the plasma vitronectin level was positively correlated with the levels of serum cholinesterase, albumin, plasma alpha 2 plasmin inhibitor-plasmin complex and the prothrombin time and results of the hepatoplastin test. Plasma vitronectin decreased with increasing severity of cirrhosis according to Child's criteria. These results suggest that the plasma vitronectin level is a useful parameter of hepatic synthetic function in patients with liver diseases; it may also reflect the severity of cirrhosis. Light microscopy revealed vitronectin in the area of focal necrosis and the portal tracts in the liver of patients with acute viral hepatitis, in the area of piecemeal necrosis in the liver of patients with chronic hepatitis and along the area of fiber deposition in the liver of patients with cirrhosis. Immunoelectron microscopy showed vitronectin in the rough endoplasmic reticulum of hepatocytes. Moreover, vitronectin was seen around inflammatory cells, endothelial cells, Ito cells and hepatocytes in the perisinusoidal area near focal necrosis and piecemeal necrosis and on collagen fibers.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of hepatitis delta virus RNA levels during interferon therapy by analysis of polymerase chain reaction products with a nonradioisotopic hybridization assay. We developed a nonradioisotopic assay for detection of hepatitis delta virus RNA in serum by combining reverse transcription of RNA, polymerase chain reaction of the resultant complementary DNA and enzyme linked immunoassay detection of the polymerase chain reaction products using a monoclonal antibody specific for double-stranded DNA. This DNA enzyme immunoassay had a limit of detection of cloned hepatitis delta virus RNA similar to that of standard PCR followed by Southern-blot hybridization (approximately 10 copies/sample) and was 10(3) to 10(4) times more sensitive than direct dot-blot hybridization (approximately 10(5) copies/sample). Serial serum samples from six patients with chronic hepatitis delta virus infection undergoing interferon therapy were analyzed by reverse transcription-polymerase chain reaction followed by both standard hybridization and DNA enzyme immunoassay. The results of both methods were comparable, revealing disappearance of hepatitis delta virus RNA after 3 to 6 mo of therapy in three patients, two of whom had also a significant decrease in ALT activity. The DNA enzyme immunoassay test is therefore a potentially useful method for therapeutic monitoring in chronic hepatitis delta virus infection and may contribute to a wider application of polymerase chain reaction in clinical laboratories.


Abnormal surface distribution of the human asialoglycoprotein receptor in cirrhosis. Serum concentrations of asialoglycoproteins are increased in cirrhosis. We hypothesized that this increase results from abnormalities in the asialoglycoprotein receptor, which is located on the sinusoidal and lateral membrane of hepatocytes. Therefore we searched for morphological alterations in the distribution of the asialoglycoprotein receptor in human liver, using a light microscopic immunoperoxidase method in autopsy livers. In 24 of 25 (96%) of patients without liver disease, the asialoglycoprotein receptor was located on the sinusoidal and, less prominently, the lateral surface of hepatocytes but not the canalicular surface. In contrast, in 12 of 18 (67%) patients with cirrhosis of various causes, the receptor also was localized strikingly along the canalicular surface, with a corresponding decrease on the sinusoidal and lateral surfaces. We conclude that an abnormal cell-surface distribution of the asialoglycoprotein receptor commonly occurs in cirrhosis. This abnormality might result in impaired clearance of desialylated glycoproteins from plasma.


Hepatocyte regeneration in acute fulminant and nonfulminant hepatitis: a study of proliferating cell nuclear antigen expression. It has been suggested that in fulminant hepatitis it is the lack of hepatocyte regeneration that in the presence of an ongoing loss of hepatocytes leads to hepatic failure and ultimately determines the grim prognosis of this disease. However, little data are available concerning hepatocyte regeneration in human acute hepatitis. We compared the nuclear expression of proliferating cell nuclear antigen with the incorporation of bromodeoxyuridine in formalin-fixed, paraffin-embedded liver tissues of rats at different stages of regeneration after two-thirds partial hepatectomy. Immunohistochemical staining for proliferating cell nuclear antigen was performed using the monoclonal antibody 19F4. A good correlation was seen between nuclear labeling for bromodeoxyuridine and proliferating cell nuclear antigen, which indicates that the immunoreactivity for proliferating cell nuclear antigen accurately reflects hepatocyte proliferation. Subsequently, we determined the nuclear expression of proliferating cell nuclear antigen on archival paraffin-embedded samples of the normal human liver (8 cases), acute nonfulminant hepatitis (10 cases) and fulminant hepatitis (4 cases). The mean proliferating cell nuclear antigen labeling indices were the following: normal liver = 0.4%; acute nonfulminant hepatitis = 43.0%; and fulminant hepatitis = 45.9%. The indices for proliferating cell nuclear antigen were significantly greater in acute hepatitis than in the normal liver, reflecting the high cell turnover in hepatitis. However, no significant difference was seen between the expression of proliferating cell nuclear antigen in nonfulminant and fulminant acute hepatitis. These data suggest that the net loss of hepatocytes in fulminant hepatitis may not be caused by a lack of hepatocyte regeneration but rather results from overwhelming hepatocyte injury with subsequent cell death.


Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells. Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1'-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.


Nested polymerase chain reaction on cellular DNA in plasma: a rapid method to investigate the collagen type I A2 MspI polymorphic restriction site in alcoholic patients. We investigated the MspI restriction site of the human collagen type I A2 (COL1A2) gene in alcoholic subjects with and without liver disease, using the nested polymerase chain reaction to analyse trace cellular DNA in plasma samples. This procedure is rapid, it requires only 200 microliters plasma, and the results correlate perfectly with those of Southern blotting. Alcoholic subjects with acute alcoholic hepatitis had a significantly lower frequency of the minor allele than healthy controls or alcoholics with minimal liver abnormalities, i.e. steatosis. Our results reinforce the hypothesis of a genetic predisposition of certain alcoholics to liver damage.


CFTR illegitimate transcription in lymphoid cells: quantification and applications to the investigation of pathological transcripts. Since the isolation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the characterization of the main mutation (delta F508) in 1989, a large number of rare mutations has been found. Full screening of the CFTR gene is difficult because it is split into 27 exons covering 250 kb of genomic DNA. This gene is essentially expressed in the lung and intestinal tract, neither of which are easily accessible for routine investigations. The recent description of a faint transcription of highly tissue-specific genes in any cell, a phenomenon known as illegitimate transcription, would facilitate the research of mutations and the characterization of truncated m-RNA caused by splicing mutations. Using the polymerase chain reaction on cDNA (cDNA-PCR), we detected transcripts of the CFTR gene in lymphocytes and lymphoblast cells at a very low level (about 300 times less than in lung or intestine). This strategy allowed us to obtain a sufficient amount of cDNA-PCR product compatible with further molecular analyses. We have, therefore, analyzed a cDNA fragment overlapping exons 10 and 11 by polyacrylamide gel electrophoresis and direct sequencing, and detected the delta F508 mutation at this level. Our protocol can be generalized to the investigation of the total 4.5-kb CFTR coding sequence.


The human vitronectin (complement S-protein) gene maps to the centromeric region of 17q. Vitronectin (complement S-protein, serum-spreading factor, epibolin) is a multifunctional glycoprotein that mediates cell-to-substrate adhesion, inhibits the cytolytic action of the terminal complement cascade in vitro and binds to several serine protease inhibitors of the serpin family, viz. antithrombin III, plasminogen activator inhibitor I (PAI-1) and II (PAI-2), heparin cofactor II and protease nexin. Using high resolution fluorescence in situ hybridization, we mapped the vitronectin gene to the centromeric region of the long arm of chromosome 17 corresponding to 17q11. The location was confirmed by co-hybridization with the centromere-specific alphoid probe p17H8 (D17Z1) and by chromosome banding with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). None of the previously mapped genes that are evolutionary related to vitronectin are located on the same chromosome.


The effect of neonatal tolerance to bovine gamma globulin (BGG) on BGG-reactive CD4+ T lymphocytes. Compelling evidence has been presented that tolerance to major histocompatibility complex (MHC) and other antigens expressed on cells of the thymus is due to clonal deletion. However, it has not been determined conclusively how tolerance to diverse antigens, which may initially interact with the immune system in the periphery, is induced and maintained. Considerable evidence exists to suggest that mechanisms other than deletion, such as clonal anergy or immunoregulation, may be involved. We have previously shown that the clonal deletion of CD4+ cells specific for bovine gamma globulin (BGG) does not occur during induction of tolerance to this soluble antigen in adult life. In this study we have extended our previous findings by examining unresponsiveness to BGG which had been established during early ontogeny, when natural tolerance of self-antigens is likely to develop. It is demonstrated here that BGG-reactive, CD4+8- T cells, which proliferate and secrete interleukin-2 on stimulation with BGG in vitro, can be obtained from mice rendered tolerant of BGG as neonates. Even though the mice from which they were derived were unable to respond to BGG, these BGG-reactive T cells, by the parameters tested here, could not readily be distinguished from the corresponding cells in BGG-immune and non-immune animals. It is therefore evident that this tolerant state is not simply the result of clonal deletion of BGG-reactive CD4+ T cells but is more likely to be due to a reversible mechanism which controls their responsiveness in vivo.


Expression and function of membrane attack complex inhibitory proteins on thyroid follicular cells. Human thyroid cells are resistant to lysis by the homologous membrane attack complex. By immunohistochemical staining we here show that normal thyroid cells and those in Graves' disease and Hashimoto's thyroiditis express two membrane attack complex-inhibiting proteins, CD59 antigen and membrane attack complex-inhibiting protein/homologous restriction factor (MIP/HRF). In vitro, the expression of both molecules was enhanced by interleukin-1 (IL-1), tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma) and cytokine-treated thyroid cells were more resistant to lysis by homologous complement. Blocking experiments with monoclonal antibodies against CD59 antigen and MIP/HRF showed that both molecules contributed but CD59 antigen was the more important in mediating resistance to complement attack. Expression of these proteins may be an important determinant of the severity of tissue injury produced by complement-fixing thyroid peroxidase antibodies in autoimmune thyroid disease.


Increased expression of highly branched N-linked oligosaccharides terminating in N-acetylglucosamine residues in neoplastic and sclerodermal chicken fibroblasts. Although neoplastic cells often show a shift towards the expression of larger N-linked oligosaccharides compared to their normal counterparts, little consideration has been given to the possibility that these changes might be a more general phenomenon characteristic of certain neoplastic and non-neoplastic proliferative disorders. Terminal N-acetylglucosamine (GlcNAc) cluster antigen (TGCA) is an immunoreactive epitope(s) of highly branched N-linked oligosaccharides terminating in GlcNAc residues. Here we have compared the expression of this antigen in normal, neoplastic and sclerodermal chicken fibroblasts by immunomorphological methods. TGCA was detectable in only a few, if any, fibroblasts of normal chicken skin or those cultured from chicken embryos. In contrast, the antigen appeared in 15 to 30% of chicken embryo fibroblasts transformed with avian sarcoma viruses and about 50% of neoplastic fibroblasts of both Rous sarcoma virus-induced fibrosarcomas and carcinogen-induced transplantable fibrosarcomas. Significantly, TGCA was also found in most activated fibroblasts in the skin of chickens with hereditary scleroderma. These results indicate that increased expression of highly branched N-linked oligosaccharides terminating in GlcNAc residues is characteristic of both neoplastic and sclerodermal chicken fibroblasts. Investigation of this phenomenon may thus provide insight into biochemical pathways involved in neoplastic transformation and pathogenesis of a number of non-neoplastic proliferative connective tissue disorders such as scleroderma. Moreover, changes in the expression of TGCA-positive oligosaccharides (or their modified biochemical counterparts in mammalian species) may have considerable value for diagnosis of several connective tissue diseases.


Staining of hyaluronan in rat cerebellum with a hyaluronectin-antihyaluronectin immune complex. The presence of hyaluronan was studied histochemically in the adult rat cerebellum. We used the hyaluronectin--antihyaluronectin immune complex technique based on the high affinity of hyaluronectin for hyaluronan. The immune complex was prepared with hyaluronectin from a human brain extract and an anti-hyaluronectin monoclonal antibody, which does not react with rat hyaluronectin. This is a specific probe for detecting hyaluronan in rat tissues without any reaction for tissue hyaluronectin. Hyaluronan was found at the nodes of Ranvier, in the perineuronal microenvironment of the deep nuclei and at the Purkinje cells surrounding the initial segment of the axon. It was located at the same places as hyaluronectin, in areas specialized in ion exchanges and neurotransmission. This suggests that the hyaluronectin-hyaluronan complex could be involved in these processes. The immune complex technique with anti-hyaluronectin monoclonal antibody thus seems to be a specific and valuable tool for investigations of the distribution of hyaluronan in the rat cerebellum.


Co-expression of vimentin and cytokeratins in M cells of rabbit intestinal lymphoid follicle-associated epithelium. Membranous epithelial (M) cells within the follicle-associated epithelium which overlies gut-associated lymphoid tissue in Peyer's patches and of appendix have been shown by immunocytochemical staining, in rabbit, to contain both vimentin- and cytokeratin-type intermediate filaments. The specificity of vimentin immunostaining has been confirmed by blocking with purified vimentin and by immunoblotting. No evidence was obtained for the expression of vimentin in rat, mouse or human M cells. The possible significance of vimentin-expression in these specialized epithelial cells and the potential use of vimentin as a positive marker for M cells are discussed.


Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa. Serotypes O2, O5, and O16 of Pseudomonas aeruginosa are chemically related, and the O antigens of their lipopolysaccharides share a similar trisaccharide repeat backbone structure. Serotype-specific monoclonal antibodies (MAbs) MF71-3, MF15-4, and MF47-4 against the O2, O5, and O16 serotypes, respectively, were isolated. MAb 18-19, which is cross-reactive with all strains of this chemically related serogroup, was also produced. When column chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated lipopolysaccharide (LPS) samples from each of the serotypes were probed with the MAbs in Western immunoblots, each of the serotype-specific MAbs interacted only with high-molecular-weight bands of the homologous LPS, with a minimum O-antigen chain length of at least 6 to 10 repeats. In contrast, cross-reactive MAb 18-19 was shown to interact in Western immunoblots with the entire LPS banding pattern except the fastest-running band, which lacks O antigen. Chemical modification of P. aeruginosa LPS by alkali treatment and carboxyl reduction abolished reactions between LPS and MAb 18-19, while reactions of modified LPS with serotype-specific MAbs were not affected. Therefore, cross-reactive MAb 18-19 likely recognizes the chemical backbone structure of the O repeat that is common to all three serotypes of the O2-O5-O16 group, while the O-specific MAbs appeared to recognize LPS epitopes that could be presented when 6 to 10 or more O-antigen repeat units are present on the LPS molecule. Thus, the O-specific LPS epitopes likely involve unique chemical structures, glycosidic linkages, and some order of folding of the O side chains.


Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA. Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis. To investigate the function of scRNA in B. subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination. Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death. The loss of the scRNA gene in B. subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA. These results provide further evidence for a functional relationship between B. subtilis scRNA, human signal recognition particle 7S RNA, and E. coli 4.5S RNA.


Detection and quantitation of sperm-bound antibodies by flow cytometry of human semen. Detection of sperm surface antibodies is important for the diagnosis and treatment of infertility. The authors have investigated the methodologic aspects of flow cytometry (FCM) to detect sperm-bound antibodies and to quantitate the sperm antibody load (antibody molecules/spermatozoa). To obtain reliable results, dead spermatozoa must be excluded from analysis because they can bind antibody nonspecifically, and comprise 10% to 58% (n = 28) of the ejaculate in subfertile men. Flow cytometry estimation of dead cells correlates (r = 0.83) significantly with the manual Eosin Y method. After staining washed sperm samples (n = 26) with fluorescein-isothiocyanate-conjugated F(ab')2 fragments of anti-IgG and IgA antibodies, the sperm load was 11,500 +/- 8,600 IgG molecules and 13,200 +/- 9,500 IgA molecules per spermatozoa. The sperm antibody load measured on different occasions could be compared between patients or in the same patient by the use of calibration standards. Since the inter- and intra-assay variation of the FCM assays was less than 10%, FCM has the potential reliably and objectively to monitor the sperm antibody load during corticosteroid treatment.


Chemical characterization of pH-dependent structural epitopes of lipopolysaccharides from Rhizobium leguminosarum biovar phaseoli. Lipopolysaccharide (LPS) was isolated from free-living Rhizobium leguminosarum bv. phaseoli CE3 cells grown at pH 4.8 (antigenically similar to bacteroid LPS) and compared with that from cells grown at pH 7.2 (free-living bacteria). Composition analysis revealed that pH 7.2 LPS differs from pH 4.8 LPS in that 2,3,4-tri-O-methylfucose is replaced by 2,3-di-O-methylfucose. The amount of 2-O-methylrhamnose is greater in the pH 4.8 LPS than in the pH 7.2 LPS. Analysis of the structural components of LPS (O-chain polysaccharide, core oligosaccharides, and the lipid A) revealed that all the composition differences in the various LPSs occur in the O-chain polysaccharide. These structural variations between pH 4.8 and pH 7.2 LPSs provide a chemical basis for the observed lack of cross-reactivity with pH 4.8 LPS of two monoclonal antibodies, JIM28 and JIM29, raised against free-living bacteria grown at pH 7.2. An LPS preparation isolated from bacteroids contained both 2,3,4-tri-O- and 2,3-di-O-methylfucose residues. This result is consistent with the finding that the two monoclonal antibodies react weakly with bacteroid LPS. It is concluded that methylation changes occur on the LPS O-chain of R. leguminosarum bv. phaseoli when the bacteria are grown at low pH and during nodule development.


Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity. We determined the location, activity, and regulation of the promoter of the Lactococcus lactis 8-kb lactose operon (lacABCDFEGX), which encodes the enzymes of the lactose phosphotransferase system and the tagatose 6-phosphate pathway. The lac promoter sequence corresponds closely to the consensus promoter described for gram-positive bacteria and is located in a back-to-back configuration with the promoter of the divergently transcribed lacR gene, which encodes the LacR repressor. The transcription start sites used under induced (lactose) and noninduced (glucose) conditions were determined. The minimal promoter region that could be isolated on a single restriction fragment included sequences ranging from -75 to +42. The effect of the presence of flanking sequences and the lacR gene on promoter activity and regulation was studied in Escherichia coli and L. lactis strains by using transcriptional fusions with promoterless chloramphenicol acetyltransferase reporter genes. The results showed that transcriptional regulation of the lac operon is mediated by the interaction between the LacR repressor, the lac promoter, and sequences in the noncoding region between the lacR and lacA genes. Sequences flanking the minimal promoter region appeared to enhance lac promoter activity much more in L. lactis (5- to 38-fold) than in E. coli (1.3- to 5-fold).


Mutations affecting translational coupling between the rep genes of an IncB miniplasmid. The nature of translational coupling between repB and repA, the overlapping rep genes of the IncB plasmid pMU720, was examined. Mutations in the start codon of the promoter proximal gene, repB, reduced the efficiency of translation of both rep genes. Moreover, there was no independent initiation of repA translation in the absence of repB translation. The position of the repB stop codon was crucial for the efficient expression of repA, with the wild-type positioning being optimal. Translational coupling was found to be totally dependent on the formation of a pseudoknot structure. A model which invokes formation of a pseudoknot to facilitate initiation of repA is proposed.


Similarity between the Myxococcus xanthus and Stigmatella aurantiaca reverse transcriptase genes associated with multicopy, single-stranded DNA. To determine the evolutional relationship of bacterial retroelements of Myxococcus xanthus and Stigmatella aurantiaca, the nucleotide sequence of 3,060 bases encompassing msr, msd, and the upstream region of msd (downstream of msr) of S. aurantiaca DW4 was determined and compared with the same region from M. xanthus. An open reading frame was found 92 bases upstream of msd which encoded a polypeptide of 480 amino acid residues having 73% identity with the reverse transcriptase of M. xanthus. Together with high homologies in msr (86%) and msd (81%) regions, the present data indicate that the reverse transcriptase genes as well as the retrons of M. xanthus (retron-Mx162) and S. aurantiaca (retron-Sa163) were derived from a common progenitor retron which possibly before the two myxobacterial species diverged.


Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product. The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.


Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening. The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains. The sequence revealed significant homology to the human thromboxane A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding. The binding was displaced with unlabeled PGs in the order of PGE2 = PGE1 greater than iloprost greater than PGF2 alpha greater than PGD2. The EP3-selective agonists, M&B 28,767 or GR 63799X, potently competed for the [3H]PGE2 binding, but no competition was found with EP1- or EP2-selective ligands. PGE2 and M&B 28,767 decreased forskolin-induced cAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA. Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.


Unfolding intermediates of the extracellular domain of the interferon gamma receptor. Reduction of proteins which require disulfide bonds to be stable in the folded state is accompanied by step-wise unfolding. A soluble human interferon gamma receptor produced in Escherichia coli was used to investigate the kinetics of formation of unfolding intermediates. The protein includes 8 cysteine residues forming four disulfide bonds. It was reduced by using either dithiothreitol or the thioredoxin reduction system. Reduction with dithiothreitol resulted in formation of mainly four monomeric unfolding species as visualized by sodium dodecyl sulfate-polyacrylamide gels. The enzymatically catalyzed reaction produced only small amounts of two monomeric products and mostly delivered oligomeric and polymeric forms. In both cases, the ligand binding capacity of the receptor was significantly reduced immediately after appearance of the first intermediate. The intermediates involved interchange of disulfide bonds and did not show ligand binding capacity. Some of them were recognized by specific antibodies which detect conformational epitopes on the native interferon gamma receptor. On the basis of the antibody binding, a preliminary characterization of the formed intermediates was attempted. When the soluble receptor was reduced in the presence of denaturing agents, the reduction products were different from the unfolding intermediates generated in the absence of denaturants.


Functional analysis of regulatory regions upstream and in the first intron of the estrogen-responsive chicken very low density apolipoprotein II gene. Estrogen regulates the expression of the yolk protein genes in the chicken liver during periods of egg laying. While all five of these genes, vitellogenins I, II, and III, very low density apolipoprotein II (apo VLDLII), and apolipoprotein B, respond to estrogen, individual controls are superimposed on their coordinate regulation with respect to the kinetics of induction, magnitude of response, and developmental expression. The estrogen-responsive Leghorn strain M hepatoma (LMH) cell line provides a model system for studying the molecular basis of the similarities and differences in the regulation of these genes. The apoVLDLII gene is regulated by estrogen in LMH cells in an appropriate time- and dose-dependent manner. Regulatory regions of the apoVLDLII gene have been identified by transient transfection studies in LMH cells. All four of the sequences previously shown to bind protein between the TAATA motif at -26 and proximal estrogen response element at -171 are essential in regulation of the apoVLDLII gene. Mutation of any single binding region reduces expression by more than 80%, indicating cooperative interactions of proteins across the entire region. While these sequences will direct assembly of a functional transcription complex, we demonstrate that addition of the first intron of the apoVLDLII gene to the promoter construct results in a 4-fold increase in estrogen-dependent expression following transient transfection into LMH cells. Results of deletion analyses indicate that two distinct regions of the intron contribute to this regulation.


Lack of agonistic activity in McN-A343 may be circumstantial. 1. In Tyrode (Ca++ 1.80 mM) or Krebs (Ca++ 3.36 mM) solution, McN-A343 evoked a dose-dependent contractile response in the naive guinea-pig ileum in the dose range 0.40-40 microM, or bladder in the dose range 0.8-200 microM. 2. After two full cycles of carbachol or bethanechol administration in Tyrode solution, neither preparation responded to McN-A343 for 45 to 60 min. The response in Krebs solution, however, was almost unaltered at 15-30 min. The rate of recovery of the contractile response appears to depend on the calcium content of the medium used. 3. A previous report that McN-A343 failed to evoke a contractile response in the guinea-pig ileum or bladder must be reconsidered in the light of the present findings.


Regional differentiation of gastric and of pyloric smooth muscle in the pig: mechanical responses to acetylcholine, histamine, substance P, noradrenaline and adrenaline. 1. Mechanical activity was recorded in isolated muscle preparations from the circular and longitudinal layers of different regions of porcine stomach and from the pyloric ring. 2. In all gastric strips acetylcholine (10(-7)-10(-4) mol l-1), histamine (10(-7)-10(-5) mol l-1) and substance P (10(-9)-10(-7) mol l-1) elicited contractions. 3. In fundic strips the catecholamines, noradrenaline and adrenaline (10(-8)-10(-5) mol l-1), produced inhibitory responses at lower concentrations and excitatory responses at high concentrations. After blockade of beta-adrenoceptors by propranolol, a pure excitatory response remained which could be inhibited by alpha-adrenoceptor blockade with phentolamine. No responses were observed in corpus and antrum. 4. Strong contractions were induced by these catecholamines in circular muscle strips of the pyloric ring, in contrast to previous findings reported for human and canine preparations. The contractile response of strips from the inner layer of the pyloric ring corresponded to an average shortening of 25-30% of resting length, at a concentration of 10(-5) mol l-1 of noradrenaline or adrenaline. The responses of preparations from the outer layer of the pyloric ring were similar to those of the inner layer but generally weaker. The excitatory responses to the catecholamines were totally suppressed by 10(-5) mol l-1 phentolamine or prazosin but remained unaltered after pretreatment with 10(-5) mol l-1 propranolol or 10(-6) mol l-1 yohimbine, which indicates that they are mediated by alpha 1-adrenoceptors. 5. Application of substance P (10(-7) mol l-1) in the pyloric preparations caused a contraction as strong as that induced by noradrenaline or adrenaline. However, histamine, which is known to induce strong contractions in canine pylorus, had--as in human pylorus--no effect on porcine pylorus at concentrations up to 10(-5) mol l-1.


Development and characterization of human anti-HBs antibodies. Peripheral blood mononuclear cells (PBMC) from 13 healthy hepatitis B vaccines were transformed with the Epstein-Barr virus (EBV) and lymphoblastoid cell lines (LCL) producing antibodies to hepatitis B surface antigen (anti-HBs antibodies). Seven LCL and two clones secreting human anti-HBs monoclonal antibody were generated and their antibodies purified. One clone was fused with a mouse myeloma and the antibody from a cloned anti-HBs secreting heterohybridoma purified. One of the 10 purified human anti-HBs antibodies was characterized as IgG4, the remainder were IgG1. The antibodies had either kappa or lambda light chains. Five of the antibodies which were conjugated to horseradish peroxidase recognised the "a" group determinants.


Use of bacterial trpE fusion vectors to express and characterize the bovine immunodeficiency-like virus core protein. The gag coding region from Bovine Immunodeficiency-like Virus (BIV) was cloned into E. coli and expressed as a bacterial fusion protein. Six different clones spanning various regions of the gag open reading frame were generated. The resulting fusion proteins were expressed at high concentrations and readily purified. A panel of bovine immune sera specifically recognized the recombinant Gag proteins, as did immune sera from animals infected or immunized with lentiviruses related to BIV, such as Equine Infectious Anemia Virus (EIAV) and Human Immunodeficiency Virus (HIV). Analysis of the deletion clones, using the bovine immune sera panel, enabled us to identify at least one major epitope which was specifically recognized by all bovine sera examined. The ease of expression, purification, and specificity of these fusion proteins should enable a thorough study of the epidemiology of BIV infection.


Use of non-neutralizing monoclonal antibodies in an ELISA for intratypic differentiation of 28 echovirus type 25 clinical isolates. Three non-neutralizing monoclonal antibodies were produced and selected against echovirus type 25 JV-4 prototype strain. They were used in an ELISA to investigate the intratypic differentiation of 28 wild isolates. Clinical isolates fell into seven different groups according to their reactivity patterns in ELISA. Two of the non-neutralizing monoclonal antibodies, 9E4 and 6D3, were highly specific, while the third, 6C9, may recognize an epitope common to other types of echoviruses. In contrast, mouse polyclonal antiserum exhibited large cross-reactivities among echovirus serotypes. The reactivity patterns and the geographical origin of the isolates were generally not correlated and, in the same area, four major antigenic variants sometimes coexisted, especially in the south of France. Moreover, reactivity patterns found with ELISA were hardly ever correlated with those observed in a previous study when neutralization tests were used. These results again underline the non-correlation between structure and biological function in the Picornavirus family.


Effect of swim-up, Percoll and Sephadex sperm separation methods on the hypo-osmotic swelling test. The separation of spermatozoa from the seminal plasma is required to prepare the spermatozoa for intrauterine insemination, in-vitro fertilization and sex selection. This study evaluated the effects of sperm preparation techniques on the functional integrity of the sperm membrane, as measured by the hypo-osmotic swelling test (HOS). Thirty-four semen specimens obtained from the male partner of infertile couples were evaluated. A semen analysis and HOS test were performed on each specimen. The remainder of the specimen was divided into three equal aliquots, the first prepared using Percoll, the second using a swim-up method and the third using a Sephadex column. After the preparation, a semen analysis and HOS test was performed on each aliquot. The mean and standard deviation for the HOS test was 72.9 +/- 8.5% initially, 71.2 +/- 13.1 after Percoll, 75.2 +/- 15.1 after swim-up and 62.4 +/- 14.5 after Sephadex. Analysis of variance showed that the mean HOS score was the same after Percoll and swim-up as it was initially but significantly lower after preparation with Sephadex. There was also a higher proportion of abnormal semen specimens (HOS less than 50%) after preparation with Sephadex than after the other preparation methods. We recommend the use of the HOS test as part of a screening panel for sperm separation.


Phospholipid binding of antiphospholipid antibodies and placental anticoagulant protein. We evaluated the interaction of antiphospholipid antibodies (aPL) with placental anticoagulant protein I (PAP I), a calcium-dependent phospholipid binding protein which may act as a natural anticoagulant. Clotting assays showed additive prolongation of clotting times with aPL and PAP I. ELISA and vesicle phospholipid binding studies showed PAP I inhibition of aPL binding to phospholipid but no inhibition of PAP I-phospholipid binding by aPL. aPL and PAP I interact additively in anticoagulant activity in in vitro clotting systems and compete for phospholipid in ELISA system. These data support the hypotheses that aPL and PAP I may recognize similar phospholipid epitopes and that in vivo interaction may occur.


Rapid detection of respiratory viruses by shell vial culture and direct staining by using pooled and individual monoclonal antibodies. The Bartels respiratory virus panel detection kit is an indirect fluorescent-antibody (IFA) method that uses pooled and individual antisera for tissue culture confirmation of seven respiratory viruses. We evaluated these reagents for detecting viral antigen in shell vial cultures and by direct staining of cells from respiratory specimens. The isolation from 254 specimens of respiratory viruses in shell vial cultures compared with standard tube cultures was highly sensitive (94%) and specific (97.3%). The numbers of viral isolates detected in three consecutive years of testing with shell vial cultures were 68 of 254 (26.8%), 101 of 381 (26.5%), and 122 of 430 (28.4%). IFA direct staining of all 1,065 specimens resulted in 183 (17.2) being uninterpretable because of inadequate numbers of cells or interfering fluorescence. The sensitivity and specificity of the interpretable IFA direct stains in comparison with shell vial cultures were 85.9 and 87.1%, respectively. For detection of 881 adequate specimens, Bartels respiratory syncytial virus IFA direct staining compared with an Ortho Diagnostics Systems direct fluorescent-antibody test for respiratory syncytial virus RSV was highly sensitive (95.5%) and specific (97%). Shell vial cultures combined with Bartels IFA reagents are a rapid alternative to standard tube cultures. Bartels IFA direct staining with individual antisera provides useful same-day screening of respiratory specimens, but the antiserum pool was not effective in screening for positive specimens because of excessive amounts of nonspecific fluorescence.


Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.


Serological markers of posttransfusion hepatitis C viral infection. Serological markers for hepatitis C virus (HCV) infection were measured in serial samples from 14 posttransfusion chronic non-A, non-B hepatitis patients by a semiquantitative dot blot immunoassay. The assay detected antibodies to HCV by use of recombinant proteins that represent putative HCV capsid (core), nonstructural protein 3 (NS3) (33c), and NS4 (c100) epitopes. Seroconversion to anti-HCV antibodies (anti-HCV) was detected in all patients. The average time to active antibody production detected by any of the recombinant proteins was 13.8 (range, 3.6 to 22.0) weeks posttransfusion or 4.6 (range, -4.5 to 13.4) weeks after the first biochemical marker of illness. Anti-HCV were detected earliest by the core antigen in most cases; however, the patterns of anti-HCV responses varied significantly among individuals. Overall, the addition of the core and NS3 antigens to the assay enabled the detection of the antibody response 4 to 5 weeks earlier than did the addition of the c100 antigen, the sole antigen used in current screening tests in the United States. Passively transferred antibodies were detected by at least one antigen in early posttransfusion samples from 12 patients and decayed below detectable levels for all antigens in only 2 patients. Antibodies to all three gene products were evident in the last sample from all five patients monitored for greater than 3 years from transfusion indicating the persistence of antibodies in patients with chronic illness. Our data show that the period following the onset of hepatitis during which anti-HCV are not detected by current screening assays can be greatly shortened by the detection of anti-HCV responses by a combination of core, NS3, and c100 antigens.


Detection of herpes simplex virus by a nonradiometric spin-amplified in situ hybridization assay. An in situ hybridization kit (Diagnostic Hybrids, Inc., Athens, Ohio) was evaluated for use in the detection and identification of herpes simplex virus (HSV) from clinical specimens. For in situ hybridization, a 10-min spin amplification onto monolayers of African green monkey kidney cells (CV-1) in 24-well polystyrene dishes, 24-h culture amplification, and hybridization with an alkaline phosphatase-labeled DNA probe were used. A total of 648 specimens were tested, including 275 specimens from patients with symptomatic diseases sent specifically for HSV detection and 373 specimens from asymptomatic immunocompromised patients sent for detection of HSV shedding. Overall, the sensitivity of the hybridization assay was 97.8% (131 of 134 specimens), with 105 of 105 (100%) specimens from symptomatic patients and 26 of 29 (89.9%) specimens from asymptomatic patients being detected. The three specimens that were false negative by in situ hybridization had low virus titers, as determined by tissue culture. The specificity was 99.6% (512 of 514 specimens). The rapid, accurate results suggest that the in situ hybridization kit may be used as an alternative to conventional tissue culture for the detection of HSV.


Species-specific identification of and distinction between Borrelia burgdorferi genomic groups by using 16S rRNA-directed oligonucleotide probes. Examination of a number of previously published aligned Borrelia 16S rRNA sequences revealed the presence of regions which could serve as oligonucleotide probe targets for both species-specific identification of Borrelia burgdorferi and distinction between genomic groups. Total cellular RNA isolated from Borrelia cultures was used in slot blot analysis. Radiolabeled oligonucleotides designed to hybridize to specific 16S rRNA targets were used as probes. These probes allowed for both species-specific identification and genomic group typing of B. burgdorferi.


High levels of Gardnerella vaginalis detected with an oligonucleotide probe combined with elevated pH as a diagnostic indicator of bacterial vaginosis. We have demonstrated a new approach to diagnosing bacterial vaginosis (BV) that is based on measuring the concentration of Gardnerella vaginalis in vaginal fluid with DNA probes. G. vaginalis is virtually always present at high concentrations in women who have BV but is also detected frequently in normal women, usually at concentrations of less than 10(7) CFU/ml of vaginal fluid. Elevated vaginal pH is another sensitive indicator of BV, although it can occur in conjunction with other conditions. We have proposed that quantitative measurements of G. vaginalis using specific DNA probes can serve as a useful aid in diagnosing BV, provided the vaginal pH is above 4.5. To test this hypothesis, a group of 113 women were first evaluated for BV by the standard set of clinical signs. Vaginal washes were collected, and aliquots were analyzed by quantitative culture for the concentration of G. vaginalis. Portions of these same samples were immobilized on nylon filters, along with standards for quantitation. The filters were incubated with a radiolabelled oligonucleotide specific for G. vaginalis 16S rRNA, and the subsequent autoradiographs were examined to determine levels of G. vaginalis in each sample. G. vaginalis at concentrations of greater than or equal to 2 x 10(7) CFU/ml and vaginal pH of greater than 4.5 were then analyzed for concurrence with the diagnoses based on clinical criteria. Results of this slot blot analysis gave a sensitivity of 95%, correctly categorizing 41 of 43 BV-positive specimens, and a specificity of 99%, correctly identifying 69 of 70 BV-negative specimens, compared with diagnosis based on clinical criteria.


Comparisons of rotavirus VP7-typing monoclonal antibodies by competition binding assay. Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.


Pharmacokinetics and fibrin specificity of alteplase during accelerated infusions in acute myocardial infarction. Pharmacokinetics and fibrin specificity of alteplase (recombinant tissue-type plasminogen activator) were determined in 10 patients with acute myocardial infarction undergoing an accelerated infusion regimen during the alteplase/anistreplase patency study (TAPS). Fifteen milligrams of alteplase was administered as an intravenous bolus injection, followed by infusions of 50 mg over 30 min and 35 mg over a further 60 min. Mean steady state plasma concentrations of alteplase during the initial 30 min were 3.2 +/- 0.84 micrograms/ml, measured immunochemically, and 2.1 +/- 0.23 micrograms/ml, measured using a functional activity assay. These values were 45% and 51% higher, respectively, than those during the standard infusion schedule (p less than 0.01). However, the predominant plasma half-life determined by model fitting based on either assay (3.3 to 3.5 min) was unaltered compared with the standard regimen. Maximal concentrations of fibrin and fibrinogen degradation products were 5.1 +/- 2.2 and 1.9 +/- 1.1 micrograms/ml, respectively. Plasminogen decreased to 70% and alpha 2-antiplasmin to 35% of values before infusion. The results indicate that 1) improved coronary patency rates during "front-loaded" infusions can be rationalized in terms of higher plasma concentrations of both free and immunoreactive alteplase, 2) kinetic variables are comparable with those of other dosing strategies, and 3) fibrin specificity is not diminished relative to that of the standard infusion regimen.


Fluent aphasia in children: definition and natural history. We compared the course of a preschool child we followed for 4 years with published reports of 24 children with fluent aphasia. Our patient spoke fluently within 3 weeks of the injury. She was severely anomic and made many semantic paraphasic errors. Unlike other children with fluent aphasia, her prosody of speech was impaired initially, and her spontaneous language was dominated by stock phrases. Residual deficits include chronic impairment of auditory comprehension, repetition, and word retrieval. She has more disfluencies in spontaneous speech 4 years after her head injury than acutely. School achievement in reading and mathematics remains below age level. Attention to the timing of recovery of fluent speech and to the characteristics of receptive and expressive language over time will permit more accurate description of fluent aphasia in childhood.