Visual evoked potentials during spontaneously occurring spike-wave discharges in rats.

Flash evoked visual potentials (VEPs) were recorded in freely moving WAG/Rij rats. These rats show spontaneously occurring spike-wave discharges in the EEG, interpreted as absence-like seizures. VEPs recorded during the presence of spike-wave discharges were compared with those obtained during normal states of vigilance as quiet wakefulness, slow-wave sleep and REM sleep. Almost similar VEPs were recorded during wakefulness and REM sleep, whereas during slow-wave sleep the second positive peak (P2) was considerably larger. In comparison with normal sleep-wake states, VEPs during spike-wave discharges showed unique changes, such as a decrease in the N1 amplitude, an increase of the P4 amplitude and an enhanced afterdischarge. Other characteristics were similar to those seen during slow-wave sleep, such as an increase of the P2 amplitude and a diminished P2-N3-P3 complex. These findings indicate sensory alterations during a spike-wave discharge. As expressed in the decrease of N1, afferent information cannot enter the thalamus during the rhythmic oscillatory mode. Such alterations may underlie the lowered responsiveness to external stimuli during spike-wave activity.


Localization of insulin-like growth factor-binding protein-5 messenger ribonucleic acid in rat ovaries during the estrous cycle.

To investigate the potential role of insulin-like growth factor-binding protein-5 (IGFBP-5) in ovarian physiology, we employed in situ hybridization and Northern analysis to localize IGFBP-5 mRNA in rat ovaries during the estrous cycle. By Northern analysis, the mRNA was abundant at all stages of the cycle. Two species of mRNAs were detected, with sizes of 6.0 and 1.8 kilobases, respectively. The relative amounts of the two transcripts changed throughout the cycle. By in situ hybridization, IGFBP-5 mRNA was expressed in only a few cell types: 1) granulosa cells of some atretic follicles, 2) some secondary interstitial cells, 3) some corpora lutea, and 4) the surface epithelium. The levels of message in both the granulosa and secondary interstitial cells changed over the cycle. At 1000 h on proestrus (before the LH/FSH surge), the message was expressed in only a few follicles. Interestingly, all were small atretic preantral (200-250 microns) follicles. At 2000 h on proestrus (after the LH/FSH surge), the IGFBP-5 mRNA was more abundant; now almost every atretic preantral follicle showed a strong hybridization signal. At 0200 and 1000 h on estrus, the mRNA appeared for the first time in granulosa cells of some atretic antral follicles and in secondary interstitial cells. Hence, virtually all atretic follicles, preantral and antral, now showed IGFBP-5 gene expression. In contrast to that on proestrus and estrus, the hybridization signal on diestrous days 1 and 2 was much less prominent and was found in only a few atretic preantral follicles. Throughout the cycle, IGFBP-5 mRNA was evident in some corpora lutea, but it was not particularly prominent. Abundant IGFBP-5 mRNA was evident in the surface epithelium, and no change was detected over the cycle. Dominant follicles were devoid of IGFBP-5 mRNA. In conclusion, this paper presents the first evidence that the IGFBP-5 gene is expressed in the adult rat ovary. The IGFBP gene is expressed in a cell-specific manner, e.g. in atretic granulosa, secondary interstitial cells, corpora lutea, and the surface epithelium, and the stage of the cycle significantly affected message levels, especially in atretic granulosa and secondary interstitial cells around estrous morning. These findings suggest that IGBP-5 may be an autocrine/paracrine regulator of ovarian physiology, particularly in relation to preantral follicle atresia.


A comparative analysis of enflurane anesthesia on primate motor and somatosensory evoked potentials.

The effect of increasing enflurane concentration on magnetic-induced myogenic cranial (Cr) and peripheral (Pr) motor evoked potentials (MEPs), and electrically induced median (MN) and posterior tibial (PTN) somatosensory evoked potentials (SEPs) was studied in 10 monkeys. MEP, recorded from abductor pollicis brevis and abductor hallucis muscles, and SEP (short- and long-latency scalp recorded potentials) variables were examined at 0.25, 0.5, 0.75, 1.0 MAC enflurane concentrations. Cr-MEPs progressively attenuated (P less than 0.01) with 0.25 MAC and were abolished (greater than or equal to 0.75 MAC) by graded enflurane concentration. Stimulation threshold for Cr-MEP was progressively elevated (P less than 0.01), and eventually reliable responses were lost (greater than or equal to 0.75 MAC). Marked scalp zone reduction to obtain Cr-MEP responses was noted with increasing enflurane concentration. Pr-MEPs and most SEP peaks maintained good replicability but showed significant amplitude reduction (P less than 0.01). MEP and SEP latency values were not significantly delayed as long as the wave form remained identifiable. We conclude that enflurane has a differential influence on Cr-MEPs and SEPs. Administration of enflurane should be discouraged while monitoring myogenic Cr-MEPs since even a subanesthetic concentration is profoundly detrimental.


Sensitive colorimetric bioassays for insulin-like growth factor (IGF) stimulation of cell proliferation and glucose consumption: use in studies of IGF analogs.

We have established two in vitro bioassay systems for quantification of insulin-like growth factor (IGF) bioactivity. The first assay was used to quantitate mitogenic activity and the second was used to quantitate metabolic activity. Both assays use BALB/c 3T3 fibroblasts grown under serum-free conditions; detection of bioactivity in assays was performed colorimetrically and did not require the use of radioisotopes. The mitogenic bioassay, which requires 48 h for detection, quantitates changes in cell number and provides an index for determining the mitogenic activity of growth factors. Changes in cell number were measured by the enzymatic reduction of exogenously added MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] to MTT-formazan by mitochondrial enzymes, which was directly correlated to cell number. The metabolic bioassay, which requires 22 h for detection, measures glucose consumption by detecting changes from the initial glucose concentration of conditioned medium after addition of various growth factors. When appropriate standards were established for these bioassays, they demonstrated a high level of reproducibility (coefficients of variation were 0.085-0.096 for the mitogenic bioassay and 0.120-0.191 for the metabolic bioassay). Both assays can be performed in 96-well microtiter plates, without the use of radioisotopes, or the limitations of conventional glucose, amino acid, or thymidine incorporation studies. In initial experiments for assay specificity, epidermal growth factor had no measurable effect in either assay. However, IGF-I, IGF-II, and insulin demonstrated effects on both metabolism and mitogenesis. In the case of the mitogenic bioassay, the maximum mitogenic activation by these growth factors was approximately 180% of control, and these factors demonstrated parallel sigmoidal dose-response curves, ranging from 0.02-2 ng/ml for IGF-I and from 2-200 ng/ml for both IGF-II and insulin. In the metabolic bioassay, in contrast to the mitogenic bioassay, insulin showed a dose-response curve whose shape was different from those of IGF-I and IGF-II. IGF-I and IGF-II stimulated glucose consumption in dose-dependent ranges of 0.02-3 ng/ml and 0.4-40 ng/ml, respectively. However, the dose-response effect of insulin was wider, ranging from 0.1-2000 ng/ml. When these assays were used to measure the bioactivity of IGF analogs, a des(1-3)-IGF-I, which has decreased affinity for IGF binding proteins, demonstrated activity equivalent to IGF-I, a [Leu27]IGF-II, which has markedly diminished affinity for the type 1 IGF receptor, exhibited approximately 0.07% of the potency of IGF-I and 1% of the potency of IGF-II.


Stimulus rate-induced VEP attenuation in preterm infants.

The effect of the stimulus rate on the single flash VEP amplitude and latency (N1) was studied in 26 mechanically ventilated, preterm infants during the first days of life. Significant decreases in VEP amplitude and increases in latency were observed within a series of 3 single flash VEPs recorded with stimulus rates of 1/4 Hz. After a recovery period of 30 sec between series, the amplitude had attained baseline level, whereas the latency delayed progressively. The underlying cause of this rate effect is speculative, as individual degrees of VEP attenuation were not statistically related to gestational age, clinical state or cerebral oxygen delivery.


Interleukin-1 alpha and tumor necrosis factor-alpha differentially regulate enkephalin, vasoactive intestinal polypeptide, neurotensin, and substance P biosynthesis in chromaffin cells.

The pattern of expression of at least four neuropeptides contained in adrenomedullary chromaffin cells is altered by exposure to the cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF alpha), alone or in combination with stimulation of other second messenger pathways. Vasoactive intestinal polypeptide (VIP) was elevated 2- to 3-fold by 1 nM IL-1 alpha within 48 h of exposure, while neurotensin and substance P synthesis were unaffected, and met-enkephalin levels were decreased 25-35%. Stimulation of VIP and substance P biosynthesis by forskolin was markedly enhanced by IL-1 alpha, while forskolin stimulation of enkephalin and neurotensin biosynthesis was unaffected. IL-1 alpha amplified the effect of phorbol myristate acetate to increase the VIP content of chromaffin cells, but antagonized phorbol ester-induced elevation of neurotensin levels. TNF alpha also demonstrated a neuropeptide-specific pattern of modulation of second-messenger effects on chromaffin cell neuropeptide levels similar to those seen with IL-1 alpha. The neuroendocrine actions of IL-1 alpha described above, unlike IL-1 action in the immune system, do not appear to be mediated through IL-2 as this cytokine did not affect VIP or enkephalin expression in the presence or absence of protein kinase stimulation. Neither IL-1 alpha nor TNF alpha affected the calcium-coupled stimulation of neuropeptide secretion and biosynthesis that occurs in response to cell depolarization in these and other neuroendocrine cells in vitro and in vivo. These data provide a functional demonstration of IL-1 and TNF receptors in chromaffin cell cultures and suggest a physiological role for cytokine production in the adrenal medulla. Since both the magnitude and direction of neuropeptide synthesis modulation by IL-1 alpha and TNF alpha are highly peptide-specific, it appears that these cytokines do not merely augment second messenger pathways that affect neuropeptide synthesis, but potentially regulate the activity of factors controlling the pattern of neuropeptide gene expression in chromaffin cells.


Chemosensory event-related potentials in man: relation to olfactory and painful sensations elicited by nicotine.

The aim of this study was to investigate the topographical distribution of chemosensory event-related potentials in relation to stimulation with nicotine. The recognition thresholds of 3 different sensations elicited by nicotine (odor, burning, stinging) were determined. Subsequently, 3 concentrations of nicotine were applied which were just above mean threshold for each of the 3 sensations. Subjects rated the intensity of odor, burning, and stinging. Additionally, they tracked the time course of these sensations. Odor and stinging appeared immediately after stimulus onset. Burning started after several seconds. Intensity ratings of burning and stinging increased with rising stimulus concentrations, whereas the odorous sensation was strongest at medium concentrations. After low and medium stimuli largest mean amplitudes were parietally obtained, whereas following stimulation with the highest concentration, amplitudes peaked at Cz.


Role of Ca2+ on vasoactive intestinal peptide-induced glucose and adenosine 3',5'-monophosphate production in the isolated perfused rat liver.

Livers from fed rats (180-240 g) were perfused noncyclically with a hemoglobin-free medium in vitro to determine whether vasoactive intestinal peptide (VIP) increases hepatic glucose production through a cAMP- or a Ca(2+)-dependent mechanism. Glucose output did not increase, but cAMP increased maximally during 10(-9) M VIP infusion. When VIP was perfused at 10(-8) M or more, glucose output increased dose dependently, whereas cAMP increased only a little during the VIP infusion, but increased greatly after the infusion. When Ca2+ was excluded from the perfusate, glucose output produced by 10(-8)-10(-7) M VIP was only 40% of that observed in the Ca(2+)-containing perfusion, and the increase in cAMP was abolished almost completely. By adding 10(-7) M A23187 for 10 min during the infusion of 10(-9) M VIP, cAMP, which increased with VIP alone, decreased during the A23187 infusion and increased again after the cessation of the A23187 infusion, whereas glucose output increased during the A23187 infusion. These results were similar to those observed with higher concentrations of VIP. When 10(-4) M isobutylmethylxanthine and 10(-8) M VIP were infused concurrently, cAMP increased rapidly during the infusion and decreased after the infusion. In conclusion, 1) glycogenolysis is produced by VIP through a Ca(2+)-dependent mechanism, rather than a cAMP-dependent one; and 2) the restriction of cAMP accumulation during the infusion of high concentrations of VIP is caused by Ca(2+)-induced phosphodiesterase activation.


Cellular generators of the cortical auditory evoked potential initial component.

Cellular generators of the initial cortical auditory evoked potential (AEP) component were determined by analyzing laminar profiles of click-evoked AEPs, current source density, and multiple unit activity (MUA) in primary auditory cortex of awake monkeys. The initial AEP component is a surface-negative wave, N8, that peaks at 8-9 msec and inverts in polarity below lamina 4. N8 is generated by a lamina 4 current sink and a deeper current source. Simultaneous MUA is present from lower lamina 3 to the subjacent white matter. Findings indicate that thalamocortical afferents are a generator of N8 and support a role for lamina 4 stellate cells. Relationships to the human AEP are discussed.


Immunocytochemical localization of estrogen and progestin receptors in the baboon (Papio anubis) uterus during implantation and pregnancy.

Although estrogen and progesterone are essential for the establishment of pregnancy in primates, localization of their specific receptors in uterine cell types during pregnancy has not been investigated. Therefore, uteri were obtained from baboons during the menstrual cycle, after steroid treatment, or during early pregnancy on days 18, 25 and 32 postovulation. Uterine and placental tissues were also collected from baboons during late pregnancy. Tissues were processed for indirect immunocytochemical localization with specific monoclonal antibodies against estrogen receptor (ER; H222) and progestin receptor (PR; JZB39). Identification of specific cell types was confirmed by iron-hematoxylin/van Gieson and Gimori's stains. Specific staining for steroid receptors was detected only in the nucleus. In the absence of ovarian steroid hormones (ovariectomized baboons), ER were present in glandular epithelium, stroma, and myometrial smooth muscle cells (SMC). In contrast, PR were absent from all uterine cell types. In the estrogen-dominated (follicular and estradiol treatment) uterus, ER and PR were detected in the nuclei of glandular and surface epithelium, stroma, and myometrial SMC. Elevated progesterone levels (luteal or after progesterone treatment) resulted in a loss of nuclear ER in stroma and epithelium, except in the deep glandular epithelium in the basalis. PR was maintained in the stroma throughout the endometrium, but was detected only in the epithelium of the deep glands. The myometrial SMC contained both ER and PR. In early pregnancy, ER was absent from the glands and stroma as early as day 18 postovulation, but was present in the wall of spiral arteries, blood vessels, and myometrial SMC. On day 18 postovulation, staining for PR was absent from all glandular epithelium, but was maintained in the stroma surrounding the glands and spiral arteries, the wall of spiral arteries, blood vessels, and myometrial SMC. Stroma away from glandular epithelium contained few PR-positive cells. This staining pattern persisted throughout early pregnancy. No apparent differences in ER and PR localization were evident between the implantation and nonimplantation sites of the endometrium and myometrium. In late pregnancy, ER were only present in the SMC of the myometrium; however, PR were detected in stroma and myometrial SMC. The maternally derived decidua expressed PR, but not ER, in the majority of cells. In contrast, fetally derived tissues, placenta, and amnio-chorion, did not contain either ER or PR at any stage of pregnancy. Clearly, ER and PR persist in particular uterine cell compartments despite the continual high levels of progesterone in pregnancy and, thus, support a receptor-mediated mechanism for estrogen and progesterone regulation of implantation and pregnancy.


Regulation of insulin-like growth factor-binding proteins in the baboon (Papio anubis) uterus during early pregnancy.

The baboon uterus begins to synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) in the deep glands of the late secretory endometrium, and this protein then becomes the major secretory product of the term decidua. We hypothesized that the placenta and/or conceptus may regulate the synthesis and secretion of IGFBP-1 by decidualized stromal cells during pregnancy. To test this hypothesis, tissue was obtained from pregnant baboons on days 18, 25, and 32 postovulation. The uterus was separated into three regions: RI (directly below the implantation site), RII (adjacent to the implantation site), and RIII (opposite the implantation site). Portions of the tissue were fixed in Bouin's solution for immunocytochemistry, and the remainder was subdivided into functionalis, basalis, and myometrium and subjected to organ explant culture. The placenta was fixed or cultured separately. Ligand blot analysis of functionalis medium showed that the major IGFBP had a mol wt (Mr) of 29,000-31,000; however, a doublet of 37,000-43,000 Mr and a band at 24,000 Mr were also present. The functionalis from all regions expressed the majority of the IGFBPs, but basalis from RI tissue also secreted the same array of IGFBPs on days 25 and 32. Ligand blot analysis of placental medium proteins revealed a doublet at Mr 37,000-43,000 on days 25 and 32, but not on day 18. Immunoprecipitation followed by ligand blot analysis of medium proteins using polyclonal antibodies to IGFBP-1 and IGFBP-2 and -3 confirmed that IGFBP-1 and -2 were the predominant products of the endometrium and decidua, while IGFBP-3 was synthesized by the placenta. Immunocytochemistry with a monoclonal antibody to IGFBP-1 demonstrated intense glandular epithelial staining in all regions on days 18, 25, and 32. Stromal staining for IGFBP-1 was first evident on day 25 and was only present in stromal cells in intimate contact with the trophoblastic tissue. By day 32, IGFBP-1 expression was not limited to the endometrial-trophoblastic junction, but extended to the deeper stromal cells and included the perivascular regions. IGFBP-1 staining was most intense in RI, but stromal cells at the luminal surface and those surrounding the spiral arteries also showed some staining in RII and RIII on day 32. These studies suggest that the baboon placenta and/or conceptus regulate IGFBP expression in the uterine endometrium during the initial stages of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)


The effect of androgen, estrogen, and growth factors on the proliferation of cultured fibroblasts derived from human fetal and adult prostates.

Stromal enlargement plays a key role in the development of benign prostatic hypertrophy in humans. Human prostatic fibroblasts were obtained from fetal and adult prostates and characterized as to their androgen and estrogen receptor status and growth in response to dihydrotestosterone (DHT), estradiol (E2), hydroxyflutamide (OH-FLU), hydrocortisone, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). In addition, the ability of hormones and growth factors to induce the messenger RNA (mRNA) for the c-fos protooncogene was assessed as a measure of the early, direct effects of these compounds on cellular proliferation. Nuclear androgen receptors were demonstrable by immunocytochemistry in both fetal and adult cells. Nuclear estrogen receptor staining was negative. Neither E2 nor hydrocortisone increased cellular proliferation. Both EGF and bFGF did increase cellular growth. DHT (10(-8)-10(-7) M) had a significant stimulatory effect on cell growth only in serum-free media. OH-FLU addition enhanced DHT induced proliferation. Changing the media during the course of the experiment obliterated the stimulatory effect of DHT. Both EGF (10 ng/ml) and bFGF (20 ng/ml) increased the mRNA for the c-fos protooncogene. DHT (10(-7) M) did not induce the mRNA for c-fos. We conclude that EGF, bFGF, and DHT (especially in combination with OH-FLU) increase the proliferation of human prostatic fetal and adult fibroblasts in vitro. E2 has no effect on fibroblast proliferation. The stimulatory effects of EGF and bFGF are direct, whereas the effect of DHT appears to be indirect, possibly mediated via the increased production and/or secretion of growth factors.


Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors.

The oestrogen receptor stimulates transcription by means of at least two distinct transcriptional activation domains, TAF-1 in the N-terminal domain and TAF-2 in the hormone binding domain. Here we show that TAF-2 activity requires a region in the C-terminus of the hormone binding domain between residues 538 and 552 in the mouse oestrogen receptor which is conserved among many nuclear hormone receptors. Point mutagenesis of conserved hydrophobic and charged residues significantly reduced ligand dependent transcriptional activation but had no effect on steroid or DNA binding. Mutation of the corresponding residues in the glucocorticoid receptor also abolished transcriptional activation. We therefore propose that the conserved region may be essential for ligand dependent transcriptional activation by other members of the nuclear receptor family.


Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities.

A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms.


A conserved 11 nucleotide sequence contains an essential promoter element of the maize mitochondrial atp1 gene.

To determine the structure of a functional plant mitochondrial promoter, we have partially purified an RNA polymerase activity that correctly initiates transcription at the maize mitochondrial atp1 promoter in vitro. Using a series of 5' deletion constructs, we found that essential sequences are located within--19 nucleotides (nt) of the transcription initiation site. The region surrounding the initiation site includes conserved sequence motifs previously proposed to be maize mitochondrial promoter elements. Deletion of a conserved 11 nt sequence showed that it is critical for promoter function, but deletion or alteration of conserved upstream G(A/T)3-4 repeats had no effect. When the atp1 11 nt sequence was inserted into different plasmids lacking mitochondrial promoter activity, transcription was only observed for one of these constructs. We infer from these data that the functional promoter extends beyond this motif, most likely in the 5' direction. The maize mitochondrial cox3 and atp6 promoters also direct transcription initiation in this in vitro system, suggesting that it may be widely applicable for studies of mitochondrial transcription in this species.


Distinct modes of transcription read through or terminate at the c-myc attenuator.

Premature termination of transcription by RNA polymerase II (pol II) occurs in the 5' region of many viral and cellular genes. Modulation of this process, or attenuation, is an important means of transcriptional control, but its mechanism is unknown. Using injected Xenopus oocytes, the efficiency of the mouse c-myc attenuator was tested when it was placed at various distances from the transcription initiation site. The attenuator functioned with each of six different pol II promoters tested; however, termination efficiency declined markedly when it was placed more than approximately 400 bases from the start site. This decline in attenuator function with distance from the start site coincided with increased sensitivity to the pol II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Thus transcription complexes situated further from the promoter appear to have a lower ability to recognize the attenuator and a greater sensitivity to DRB. Furthermore, polymerases which have read through one attenuation site have a reduced ability to terminate at a second site. The results imply that a discrete subset of elongation complexes is capable of premature termination, and that this subset exists only within the first few hundred bases of the transcription unit. Regulation of termination efficiency may be effected by changing the balance between the two modes of transcription committed either to read through or to terminate prematurely.


Domain structure of the human immunodeficiency virus reverse transcriptase.

The spatial arrangement of subunits p51 and p66 of the HIV-1 reverse transcriptase and the position of the RNase H containing domain, p15, have been determined by means of neutron small-angle scattering. The reverse transcriptase (p66/p51) is a flat molecule, which can be approximated by an ellipsoid with the half axes of 5.2 nm, 4.8 nm and 1.4 nm. The two subunits p51 and p66 having a centre-to-centre distance of 3.3 +/- 0.3 nm are attached at their flat sides, slightly shifted sideways. The p15 domain is located at the long axis of the ellipsoidal reverse transcriptase having a distance of 5.0 +/- 0.5 nm to the centre of the p51d domain, which is part of the p66 subunit, and a distance of 5.3 +/- 1.2 nm to the centre of the neighbouring p51s subunit.


Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis.

The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA. The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA. This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo. Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo. A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex'). The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis. Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing. An in vitro analysis supported the same conclusion. These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required. The implications of these findings are discussed.


An archaebacterial terminal oxidase combines core structures of two mitochondrial respiratory complexes.

The operon coding for a respiratory quinol oxidase was cloned from thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It contains three genes, soxA, soxB and soxC. The first two genes code for proteins related to the cytochrome c oxidase subunits II and I, respectively. soxC encodes a protein homologous to cytochrome b, which is a subunit of the mitochondrial and bacterial cytochrome c reductases and the chloroplast cytochrome b6f complex. soxA is preceded by a promoter and the genes are cotranscribed into a 4 kb mRNA. Their protein products form a complex which has been partially purified and has quinol oxidase activity. The reduced minus oxidized absorption spectrum of the complex has two maxima at 586 and 606 nm. The latter is typical of cytochrome c oxidase. The complex contains four haems A. Two haems belong to the 'cytochrome oxidase' part of the complex and two are probably bound to be apocytochrome b (SoxC) and responsible for the 586 nm absorption peak. The homology between the sox gene products and their mitochondrial counterparts suggests that energy conservation coupled to the quinol oxidation catalysed either by the Sulfolobus oxidase or two mitochondrial respiratory enzymes may have a similar mechanism.


Isolation of a hagfish gene that encodes a complement component.

It has been widely accepted that cyclostomes are the most primitive vertebrates extant with the ability to produce antibodies. We isolated cDNA clones that encode a putative 'antibody' from one of the cyclostomes, Eptatretus burgeri. The amino acid sequence predicted from the nucleotide sequences of the cDNA clones indicated that this gene does actually encode the proteins isolated as hagfish 'antibodies' by various investigators. However, these proteins are not similar to mammalian immunoglobulins but have some characteristics common to complements C3, C4 and C5 in higher vertebrates. We discuss the relationships of the isolated gene for hagfish complement with the mammalian genes for complements C3, C4 and C5. We also discuss the possibility of the presence of antibodies in cyclostomes.


Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c-src we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c-src is detergent-soluble in resting platelets, while 40% of total platelet pp60c-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.


Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.


Calcium influx through subunits GluR1/GluR3 of kainate/AMPA receptor channels is regulated by cAMP dependent protein kinase.

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by three major classes of glutamate receptors, namely the ionotropic NMDA (N-Methyl-D-Aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid) receptors and the metabotropic receptor type. Among the ionotropic receptors, NMDA receptors are thought to mediate their physiological response mainly through the influx of extracellular calcium, while KA/AMPA receptor channels are mainly thought to carry the influx of monovalent cations. Recently, we have challenged this view by showing that cloned KA/AMPA receptor subunits GluR1 and GluR3 form ion channels which are permeable to calcium. We now directly demonstrate large increases in intracellular calcium concentrations induced by calcium fluxes through KA/AMPA receptor channels in solutions with physiological calcium concentrations. Calcium fluxes were observed through glutamate receptor channels composed of the subunits GluR1 and GluR3, which are both abundantly present in various types of central neurones. The calcium influx was fluorometrically monitored in Xenopus oocytes injected with the calcium indicator dye fura-2. Bath application of the membrane permeable analogue of adenosine cyclic monophosphate (cAMP) potentiated the current and also the flux of calcium through open KA/AMPA receptor channels. Further pharmacological experiments suggested that this effect was mediated by the activation of protein kinase A. Our results provide a molecular interpretation for the function of calcium permeable KA/AMPA receptor channels in neurones and identify two of the subunits of the KA/AMPA receptor channel which are regulated by the cAMP dependent second messenger system.


Gene transfer experiments imply instructive role of major histocompatibility complex class I molecules in cellular peptide processing.

DBA/2-derived mouse tumor cells were transfected with the H-2 Kb gene. Naturally processed minor histocompatibility (H) peptides were extracted from both transfected and non-transfected cells by acid elution, and were separated by high-performance liquid chromatography. Kb-restricted minor H epitopes corresponding to H-4b and mapki, both encoded by non-major histocompatibility complex genes of DBA/2, were readily detected by the respective cytotoxic T lymphocyte in peptides extracted from Kb-transfected, but not from non-transfected or Db-transfected cells. Titration experiments indicated at least 3000-fold less copies of correctly processed Kb-restricted epitopes in cells without Kb as compared to cells with Kb. Since we estimate the copy number of Kb-restricted H-4b epitopes in Kb-expressing transfectants to be less than 1000 per cell, the pool size of H-4b epitopes correctly processed in the absence of Kb should be less than 1/3 copy per cell.


Modulation of homeobox gene expression alters the phenotype of human hematopoietic cell lines.

We have previously reported that certain genes of the HOX2 cluster of homeobox genes on human chromosome 17 are specifically expressed in human leukemic cell lines with erythroid potential, suggesting that these genes are involved in hematopoietic differentiation. We now show that the expression of the HOX 2.2 gene decreases during erythropoietin-induced differentiation of the erythroid cell line MB02. In order to study the role of the HOX 2.2 homeobox gene in hematopoiesis, vectors producing sense or antisense transcripts were introduced into K562 and HEL cells, pluripotent lines with erythroid and myeloid features. Overexpression of HOX 2.2 is associated with loss of erythroid features in both lines and an increase in certain myelomonocytic markers in K562 cells. Expression of antisense HOX 2.2 is associated with an increase in erythroid features in HEL cells and a mild decrease in myeloid characteristics in K562 cells. Overexpression of the adjacent HOX 2.1 gene in K562 cells does not produce similar phenotype changes. These data demonstrate that modulation of a specific HOX 2 homeobox gene can change the phenotype of somatic cells and suggest that certain HOX 2 genes play a role in blood cell differentiation.


The effect of an immunoglobulin mu transgene on B cell maturation.

In mu 17.2.25-transgenic (M54) mice the absolute number of surface IgM (sIgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a mu transgene on B cell development, adoptive cell transfers were performed using mu transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM-positive B cells reconstituted by transgenic bone marrow in allotype-matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype-congenic and neonatal SCID recipient mice. The results indicate that the presence of a complete mu heavy chain transgene does not result in a selective developmental block of "conventional" bone marrow-derived pre-B and B cells.


Diversity of T cell receptor alpha and beta chain genes expressed by human T cells specific for similar myelin basic protein peptide/major histocompatibility complexes.

T cell receptor (TcR) alpha and beta nucleotide sequences involved in the human autoreactivity to myelin basic protein (MBP) were studied by screening cDNA libraries derived from 11 independent T lymphocyte clones (TCC) established from multiple sclerosis patients and healthy donors. The TCC with defined MBP peptide specificity and HLA-DR restriction expressed multiple TcR. Even TCC recognizing the same human MBP peptide [amino acids (aa) 139-153] in identical or very similar HLA-DR context expressed diverse TcR. Two TCC which recognized peptide aa 139-153 equally well in the context of both HLA-DR2a and -DR1 molecules used distinct TcR alpha but identical beta chains. The knowledge of TcR beta and TcR alpha chain sequences of human MBP-specific T cells will allow studies correlating structure and function of TcR and their targets in MBP autoreactivity. This may have an impact on the development of immunotherapies in multiple sclerosis.


Constraints in antigen processing result in unresponsiveness to a T cell epitope of hen egg lysozyme in C57BL/6 mice.

T cell hybridoma clones were derived after fusion of BW-5147 parent cells with lymphocytes from C57BL/6 mice injected with phosphorylcholine (PC)-hen egg lysozyme (HEL) conjugates. Several T cell hybridomas were preferentially reactive with PC-HEL over unconjugated HEL, and a particular clone (PC-H4.1) was further analyzed. This T cell hybridoma clone could respond to its maximal level toward unconjugated HEL only when the dose of HEL was increased to 5-10-fold of the PC-HEL concentration. Interestingly, this clone was not stimulated by unfolded HEL (by S-carboxymethylation) to the level of PC-HEL. A synthetic peptide representing the amino acid position 47-61 of HEL, which is known to be non-immunogenic upon HEL injection in C57BL/6 mice, was able to stimulate the hybridoma only to a level comparable to that induced by unconjugated HEL. The T cell response to this synthetic peptide required an additional antigen-processing step, based on its inability to stimulate T cells after treatment of antigen-presenting cells with leupeptin, chloroquine or paraformaldehyde. Deletion of a single C-terminal amino acid residue of HEL 47-61 (arginine) significantly enhanced (10-100-fold of HEL 47-61) the T cell reactivity and abrogated the necessity of further antigen processing. These results suggest that the lack of a T cell response to a certain epitope may not be due to the lack of a T cell repertoire reactive to the epitope. In some cases, the unresponsiveness may be due to the difficulty in generating the particular epitopes. Taken together, modification of the lysozyme molecule with PC conjugation may facilitate further antigen processing of HEL to generate an optimal epitope for the nonresponder mice.


CD59 expressed by human endothelial cells functions as a protective molecule against complement-mediated lysis.

CD59 is a 18-20-kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement (C) on homologous cells. In the present study we analyzed the expression and function of CD59 on human endothelial cells. Immunohistochemical analysis of renal cortex demonstrated a predominant expression of CD59 on peritubular capillary endothelial cells and glomerular endothelial cells. Flow cytometry analysis showed that human umbilical vein endothelial cells (HUVEC) expressed CD59 and the fluorescence intensity was approximately four times that of peripheral blood lymphocytes. CD59 is detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single 20-kDa molecule in 2% deoxycholate extracts of HUVEC. CD59 was released from the surface of HUVEC by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor. The functional activity of CD59 expressed on HUVEC was studied. Blocking of CD59 antigen with F(ab')2 fragments of polyclonal anti-CD59 enhanced markedly the susceptibility of HUVEC to C-mediated lysis. This effect was dependent on the amount of blocking antibodies added. Northern blot analysis revealed the presence of three species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for CD59, with sizes of about 800, 1400 and 2000 bp. These findings suggest that CD59 may be important in protection of endothelial cells against C-mediated damage at local sites of inflammation, thereby maintaining the vascular integrity in vivo.


Regulation of L-selectin expression on cultured bone marrow leukocytes and their precursors.

L-selectin (LECAM-1, LAM-1, MEL-14 antigen, Dreg antigen) is one of the molecules controlling lymphocyte homing from the blood to peripheral lymph nodes and granulocyte adhesion to inflamed endothelium. In this work, regulation of L-selectin expression on mouse bone marrow cells was studied. L-selectin-negative cells were isolated by panning technique, cultured for 1-7 days with cytokines and mitogens, and L-selectin expression was analyzed by immunofluorescence staining. When cultured for 3 days with interleukin (IL) 1, IL 2, IL 5, IL 6, phytohemagglutinin, pokeweed mitogen or in the medium alone, 75%-85% of L-selectin-negative large cells (including granulocytes, macrophages/monocytes, blasts and their precursors) became L-selectin positive. In contrast, IL 3, IL 4, granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) prevented the induction of L-selectin in a time- and dose-dependent manner. GM-CSF was the most potent inhibitor and only 10%-15% of cells became L-selectin positive after 3 days of culture. Furthermore, L-selectin was down-regulated on cultured unselected bone marrow cells by IL 3, IL 4, GM-CSF and LPS stimulation. After culture, the relative molecular mass of L-selectin was 100 kDa, similar to the size of the granulocyte form of this antigen. Cultured cells adhered to high endothelial venules (HEV) only 10%-32% as effectively as freshly isolated bone marrow cells despite high levels of L-selectin expression. The phenotypic analysis and the HEV binding data indicate that after culturing L-selectin was almost exclusively expressed on bone marrow leukocytes of myeloid series, and on these cells it was not functional in mediating peripheral lymph node HEV binding. Overall, these results show that the expression of L-selectin can be modulated by regulating the maturation and differentiation of the cells in vitro. This supports the idea that different cytokines and mitogens may also be important in controlling migrational status of leukocytes in vivo.


Existence of mature human CD4+ T cells with genuine class I restriction.

A human T cell receptor (TcR) alpha/beta CD4+CD8-T cell clone (R416) is reactive with the minor histocompatibility antigen H-Y in the context of major histocompatibility complex (MHC) class I and not class II molecules. Therewith clone R416 violates the so-called specificity association of mature TcR alpha/beta+ T cells. R416 displays H-Y-specific, HLA-A2-restricted proliferation as well as cytotoxicity in vitro. Its fine specificity is identical to that of a classical H-Y-reactive CD4-CD8+ MHC class I-restricted CTL clone, showing that CTL expressing either CD4 or CD8 can display identical antigenic specificities. Exploiting the MHC class I restriction of this CD4+ T cells clone, it was found that interaction of CD4 with non-TcR-bound MHC class II molecules does not contribute to antigen specific activation of these CD4+ T cells. This coreceptor-mismatched T cell clone was not generated in vitro but obtained by expansion of CD8-depleted peripheral blood mononuclear cells of a female who had been immunized against H-Y. The existence of such MHC class I-restricted mature TcR alpha/beta+ T cells expressing CD4 and not CD8 is relevant because it indicates that the generally accepted model for thymic selection, in which the TcR specificity alone determines CD4/CD8 expression of mature thymocytes, may not be absolute.


Ribosomal RNA gene patterns of Helicobacter pylori from surgical patients with healed and recurrent peptic ulcers.

Fifty-two strains of Helicobacter pylori were examined by DNA restriction endonuclease digestion, ribosomal (r)RNA gene probe hybridization and biotyping. Most (49) strains originated from gastric (antral) biopsies of patients before or after elective surgery for duodenal ulcers. Chromosomal DNA Hind III ribopatterns showed 9 strain clusters of which the largest contained 12 strains each with 3 common bands (1.50, 3.45, and 4.26 kb) but which were heterogeneous with respect to biotype and total digest pattern. Isolates from post-operative patients with either healed or recurrent ulcers showed ribopattern heterogeneity and exhibited a similar distribution of H. pylori ribopattern types; no single type predominated in any patient group or was more highly associated with recurrent ulcers than with healed ulcers. Multiple isolates from two surgical patients had only minor genomic variations in each set whereas isolates from two brothers had different ribopatterns. We conclude that Hind III ribopatterns in conjunction with total digest patterns might provide the basis for future epidemiological typing studies.


Choroidal vascular repair: scanning and transmission electron microscopy.

Repair of the choroidal vasculature following laser photocoagulation in the rat was examined with vascular casts and correlated with observations on thin-sections. The regenerative process began at the periphery of the damaged area, starting from the surviving choriocapillaris and venules, and proceeding towards the center by means of recanalization of damaged vessels and growth of new ones. In small healed lesions the capillary bed was re-formed. It resembled the adjacent undamaged choriocapillaris morphologically but appeared to be less dense than the intact choriocapillaris when examined by scanning microscopy. In large lesions the capillary bed was re-formed at the periphery but not at the center. Also present at the edges of the large lesions were groups of new vessels which, when observed by scanning microscopy, appeared to extend in two directions; towards the subretinal space and towards the choroidal network. Another aspect of the repair process was the simultaneous occurrence of new vessel growth and capillary regression, which was observed both at the level of the choriocapillaris and at the foci of new vessels.


Evaluation of the hepatotoxicological effects of a drug in an in vivo/in vitro model.

Both in vivo and in vitro models have certain disadvantages for the study of the chronic hepatotoxicity of drugs. The aim of this work was to evaluate a new approach based on an in vivo/in vitro model. After chronic in vivo treatment of rats with Vincamine and Vindeburnol (an eburnamenine derivative which exhibits hepatotoxic properties in man) liver cells were isolated, and functional and metabolic disorders (metabolic utilization of fructose and protein biosynthesis) were studied to determine injury. The results showed no modification of blood parameters, but a direct relationship between the dose of Vindeburnol administered in vivo and the metabolic disorders observed in vitro, evidencing the high sensitivity and reliability of this model.


Correlation between car ownership and leukaemia: is non-occupational exposure to benzene from petrol and motor vehicle exhaust a causative factor in leukaemia and lymphoma?

Although there is widespread agreement that many cancers have environmental causes we are often unable to see associations between specific cancers and exposure to environmental chemicals. One might also speculate that the more widespread, common-place and 'normal' a chemical exposure is perceived to be then the less likely it will be that the exposure is recognised, let alone be considered to cause cancer. Widespread contamination of air by chemicals associated with internal combustion may be an example of one such 'invisible' carcinogenic exposure. Yet evidence is available which suggests that many leukaemia and lymphoma cases, as well as other cancers, may be caused by this mundane and ubiquitous environmental contamination. The hypothesis is developed that leukaemia 'clustering' as well as national leukaemia incidence may be related to non-occupational exposure to benzene formed by petrol combustion and resulting from petrol evaporation. The possible association between exposure to fuel vapours, internal combustion products and cancer merits much closer examination than it receives at present.


Characterization and applications of the disc angiogenesis system.

A model to study microvascular proliferation, the Disc Angiogenesis System (DAS), consists of a synthetic foam disc implanted subcutaneously in experimental animals. After a period of growth, usually 7 to 21 days, the disc is removed. Planar sections are used to measure and characterize the growth. Microvessels grow centripetally into the disc, together with fibroblasts. Concentric growth zones have been defined by light and electron microscopy. Moderate growth occurs spontaneously and is accelerated by angiogenic stimulants placed in the center of the disc. Morphometric analyses have shown that vessel growth is directly proportional to total fibrovascular growth, so the former can be quantified by procedures measuring the latter. These include manual projection of sections and computer-assisted digital image analysis, which is recommended for routine use. The proliferation of endothelial and other cells is determined by incorporation of tritiated thymidine, using scintillation counting and autoradiography. Using the DAS, well-established angiogenic agents such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and prostaglandin E1 were found to increase proliferation of endothelial cells (EC) and microvessels. Heparin augmented the effect of bFGF. When used by itself heparin increased angiogenesis but not EC proliferation, in keeping with in vitro observations indicating that it stimulates migration but not proliferation of EC. Locally applied hyperthermia and ionizing radiation decreased angiogenesis, even when applied after the angiogenic stimulus. Systemic prostaglandin synthetase inhibitors antagonized the angiogenic effects of bFGF and EGF, in accordance with a postulated role of prostaglandins in the transduction of proliferative signals in microvascular EC. The DAS is easy to assemble and implant in small animals, including mice, which tolerate it well. Hence multiple discs can be used for each time or dose point, which allows reproducible measurements of vascular growth and increases statistical accuracy. Another advantage of the system is the capability of discriminating between proliferation and migration of EC and fibroblasts. The DAS can be used to test putative agonists or antagonists of angiogenesis. More generally, the DAS provides a model of wound healing, either uncomplicated or complicated by inflammation, and of angiogenic responses to solid tumors.


Galanin reduces PDBu-induced protein phosphorylation in rat ventral hippocampus.

The effect of galanin (GAL) on basal and phorbol-12,13-dibutyrate (PDBu) induced protein phosphorylation in rat ventral hippocampal miniprisms was investigated. GAL (0.5, 1 and 2 microM) inhibited PDBu stimulation in a concentration-dependent manner without altering basal protein phosphorylation. This inhibitory effect was prevented by the GAL antagonist galantide. GAL did not affect either the activity of protein kinase C (PKC) from rat brain or basal phosphorylation in ventral hippocampal hippogenates, suggesting that it did not directly modulate PKC activity. Depolarization of miniprisms from ventral hippocampi by 18 mM K+ prevented the effect of GAL on PDBu-induced phosphorylation. The results indicate that GAL indirectly regulates neuronal protein phosphorylation by a GAL receptor-mediated action.


Structure and RNA content of the prosomes.

Duck erythroblasts prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of approximately 720,000 +/- 50,000, a radius of gyration of 64 +/- 2 A and a hydrodynamic radius of approximately 86 A were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 A, a height of 170 A and a diameter of 40 A for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.


RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms.

A method for the rapid preparation of a defined substrate to monitor RNase H activity has been developed. Using this substrate, we have investigated the RNase H activities of the different forms of recombinant HIV-1 and HIV-2 reverse transcriptase (RT) in detail. As we report here, RNase H activity is associated only with the dimeric forms (p51/p66 or p66/p66) of the enzymes.


Divergent ileal IGF-I and IGFBP-3 gene expression after small bowel resection: a novel mechanism to amplify IGF action?

Changes in the levels of ileal insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) mRNA in the rat following massive small bowel resection (MSBR) have been investigated with a sensitive S1 nuclease assay. IGF-I mRNA levels vary little over 7 days; in contrast IGFBP-3 mRNA levels decreased to one-third 7 h post-MSBR, and remained suppressed for the length of this study. We postulate that decreased ileal synthesis of IGFBP-3 enhances the ability of IGF-I to stimulate the adaptive response.


Effects of dexamethasone on the growth and epidermal growth factor receptor expression of the OVCA 433 ovarian cancer cells.

We studied the correlation between dexamethasone (Dex) induced growth effects and modulation of epidermal growth factor receptor (EGFR) expression in OVCA 433 ovarian cancer cells. These cells express specific high and low affinity 125I-EGF binding sites and are growth stimulated by EGF. Dex exhibits mitoinhibitory effects by recruiting OVCA 433 cells in the G0-G1 phase of the cycle, but increases the number of both the high and the low affinity EGFR in a dose dependent manner. The maximal EGFR expression increase occurs after 24 h of Dex treatment consistently with Northern blot studies. The mitogenic activity of EGF in OVCA 433 cells is not affected by the presence of Dex. Moreover Dex growth inhibition occurs in JA1 cells, an ovarian cancer cell line which expresses unfunctional EGFR and which is unresponsive to EGF. Our results indicate that the Dex induced growth effects occur independently of EGFR expression.


Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.

The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.


Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands.

Expression of the int-3 locus is activated in mouse mammary tumors as a consequence of insertional mutagenesis by the mouse mammary tumor virus (MMTV). Integration of the MMTV provirus into the int-3 locus promotes the transcription and translation of flanking cellular int-3 sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila, and with the yeast cell cycle regulatory genes cdc10 and SWI6. To determine the in vivo consequences of activated int-3 expression, transgenic mice were generated harboring a genomic tumor DNA fragment consisting of the MMTV LTR and the flanking cellular int-3 sequences. All six int-3 founder transgenic mice and the progeny of one established line exhibited similar dramatic phenotypic abnormalities in tissues in which the transgene was expressed. Focal and often multiple poorly differentiated mammary and salivary adenocarcinomas appeared in the majority of transgenic mice between 2 and 7 months of age. Significantly, mammary glands were arrested in development and were lactation deficient in all female int-3 mice. The salivary glands, glands of the nasal mucosa and maxillary sinus, the extraorbital lacrimal glands, and the Harderian glands of juvenile and adult transgenic mice all contained proliferating immature ductule cells and were incompletely differentiated. In addition, all male int-3 transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis. These findings demonstrate in vivo that expression of the activated Notch-related int-3 gene causes deregulation of normal developmental controls and hyperproliferation of glandular epithelia.


csgA expression entrains Myxococcus xanthus development.

The development cycle of the myxobacterium Myxococcus xanthus consists of three partially overlapping morphological stages referred to as rippling, fruiting body formation, and sporulation, all of which are absent in csgA null mutants. The CsgA gene product is an extracellular protein, referred to as the C signal, which is essential for developmental cell-cell interactions. csgA expression increases throughout development, reaching its peak during sporulation. CsgA was made limiting for development by constructing nested deletions upstream from the csgA gene, which resulted in reduced csgA expression. Successively larger deletions resulted in termination of development at earlier and earlier stages, with rippling requiring approximately 20% maximum csgA expression, fruiting body formation requiring approximately 30% expression, and sporulation requiring 82% expression. Conversely, artificial induction of csgA also induced development provided nutrients were limiting. These results suggest that steady increases in CsgA over the course of development entrain the natural sequence of morphological events. The csgA upstream region appears to process information concerning the levels of nutrients, peptidoglycan components, and the B signal. In the absence of nutrients, a region extending 400 bp upstream from the start site of transcription was necessary for development and maximal csgA expression. In the presence of low levels of nutrients, a region extending approximately 930 bp upstream was essential for the same tasks. It appears that the upstream region extending from -400 to -930 stimulates csgA expression in the presence of excess carbon, nitrogen, and phosphate, thereby allowing development to go to completion.


The Rex system of bacteriophage lambda: tolerance and altruistic cell death.

The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.


Analysis of a ribosomal RNA operon in the actinomycete Frankia.

The organisation of ribosomal RNA-encoding (rrn) genes has been studied in Frankia sp. strain ORS020606. The two rrn clusters present in Frankia strain ORS020606 were isolated from genomic banks in phage lambda EMBL3 by hybridization with oligodeoxyribonucleotide probes. The 5'-3' gene order is the usual one for bacteria: 16S-23S-5S. The two clusters are not distinguishable by restriction enzyme mapping inside the coding section, but vary considerably outside it. Sequencing showed that the 16S-rRNA-encoding gene of ORS020606 is very closely related to that of another Alnus-infective Frankia strain (Ag45/Mut15) and highly homologous to corresponding genes of Streptomyces spp. Two possible promoter sequences were detected upstream from the 16S gene, while no tRNA-encoding gene was detected in the whole operon. Regions with a high proportion of divergence for the study of phylogenetic relationships within the genus were looked for and found in the first intergenic spacer, in the 23S and in the 16S gene.


Interaction between viruses and monoclonal antibodies studied by surface plasmon resonance.

An automated biosensor system designed for measuring molecular interactions in real time and without any labelling of the reactants has been used to study the interaction of two animal viruses (vaccinia virus and poliovirus) and two plant viruses (cowpea mosaic virus and tobacco mosaic virus) with monoclonal antibodies. Using monoclonal antibodies specific for different conformational states of viral protein, it was found that the virus particles retained their conformational integrity when immobilized on the dextran matrix present on the sensor chip. Compared to conventional solid phase immunoassays, in which immobilized proteins are usually partly denatured, the biosensor system presents several advantages for studying virus-antibody interaction.


A crude extract of Artocarpus integrifolia contains two lectins with distinct biological activities.

The crude extract derived from seeds of Artocarpus integrifolia (jack fruit) contains two fractions with different biological activities for lymphocytes. One fraction is the D-galactose-binding lectin, jacalin, obtained by affinity purification on a D-galactose agarose column. The other, which is a component of the flow-through fraction (FT), is responsible for the mitogenic activity observed with human PBMC and murine spleen cells. In contrast, jacalin inhibits FT- and ConA-induced proliferative activity of human PMBC and murine spleen cells. This inhibition is not due to toxicity, because: (1) jacalin induces significant levels of IL-3/GM-CSF but not of IL-2 and/or IL-4 in murine spleen cells; (2) jacalin does not affect the capacity of these cells to secrete IL-2 or IL-4 as supernatants obtained from spleen cells sequentially stimulated with jacalin and ConA contain IL-2 and/or IL-4 as well as IL-3/GM-CSF. The ligand for the mitogen contained in the FT fraction is D-mannose as determined by sugar inhibition studies.


Cell-free CD5 in patients with rheumatic diseases.

Since an increased frequency of CD5+ B cells has been reported in rheumatoid arthritis (RA) and primary Sjögren's syndrome (SS), and the expression of the molecule was reduced on the T cells of some SS patients, we hypothesised that there would be an accelerated turnover of CD5 in these disorders. We describe a novel enzyme-linked immunosorbent assay for measuring cell-free (CF) CD5, using rabbit F(ab')2 anti-CD5 antibody as capture agent and monoclonal anti-CD5 antibody as revealing agent. It was established that CF-CD5 was detectable in RA and SS sera, as opposed to sera from patients with ankylosing spondylitis and normal controls. The level of CF-CD5 did not correlate with rheumatoid factor in RA patients but was significantly higher (P less than 0.05) in SS patients with extraglandular manifestations than in those with glandular disease. Three of the latter patients with significantly increased levels of CD5-negative T cells did not have a particularly high proportion of CF-CD5 in these sera.


Activation of human natural killer cells by the protein-bound polysaccharide PSK independently of interferon and interleukin 2.

The protein-bound polysaccharide PSK was tested for the ability to activate human natural killer (NK) cells. When blood lymphocytes and purified CD3-CD16+ large granular lymphocytes (LGL) were treated in vitro overnight with PSK, they demonstrated enhanced NK cell activity against K562. The PSK-activated killer cells also lysed NK-resistant targets and freshly isolated autologous and allogeneic tumor cells. The PSK effect was observed with concentrations that could be obtained in the blood of cancer patients receiving oral administration of PSK. PSK-induced enhancement of NK activity was not abrogated by monoclonal antibodies (mAb) that neutralized interferon (IFN) alpha, IFN gamma, or interleukin-2 (IL-2). In addition, mAb reactive with p55 (alpha chain) or p75 (beta chain) glycoproteins of IL-2 receptors had no effects on PSK-enhanced NK activity even when used simultaneously. These results indicate that the PSK could activate human NK cells independently of IFN and IL-2/IL-2R systems.


Expression of CD54, CD58, CD14, and HLA-DR on macrophages and macrophage-derived accessory cells and their accessory capacity.

Human peripheral monocytes can differentiate in vitro into macrophages (Mph) possessing a low accessory activity in T cell stimulation. Mph can be converted into a state of high accessory activity by treatment with dibutyryl cyclic AMP. This finding was used in this study to achieve Mph-derived AC (MphAC). Among the surface antigens on AC which have been shown to participate in accessory events leading to T cell proliferation, MHC class II antigens, CD58 (LFA-3) and CD54 (ICAM-1) seem to be especially important. We show here that the high accessory capacity of MphAC was not correlated with a high level of the surface antigens HLA-DR, CD58, and CD54. The amount of CD54 molecules was, in fact, lower on the MphAC than on the Mph.


Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus.

The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses--IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of Mr (approx. 41,000 to less than 12,400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus that may protect bacteria against the defence mechanisms of the host.


Molecular cloning of the bovine thymopoietin gene and its expression in different calf tissues: evidence for a predominant expression in thymocytes.

Thymopoietin (TP), a 49 amino acid peptide, is regarded as a thymic hormone, secreted specifically by some epithelial cells in the thymic stroma and exerting a multitude of effects on maturation and function of T lineage cells. As part of our study on the molecular biology of TP, we isolated cDNA clone coding for a bovine TP precursor and used it as a probe to analyze the presence of mRNA coding for TP in different tissues. The cDNA clone reported here is 1.1 kb long and contains an open reading frame (ORF) of 741 bp which corresponds to 247 amino acids. The 147 bp coding for the entire bovine TP are at the 5' end of the ORF. A DNA fragment coding for amino acids 1-42 of bovine TP was used as a probe to look for hybridizable RNA sequences, extracted from various calf tissues, by the S1 nuclease protection method. Our results indicate that the TP gene is expressed predominantly in lymphatic tissues. Lymphatic tissues with the highest levels observed were thymocytes and not thymic stroma. Lower, but still significant, amounts were present in tonsils, neck lymph nodes, and small intestine (probably because of its lymphatic part--the Peyer's patches), whereas cultured thymic stromal cells, spleen tissue and peripheral blood mononuclear cells displayed a low level of TP mRNA. The TP gene expression in all other (non-lymphatic) tissues tested, was weak, barely detectable or virtually absent. However, the cerebellum could be singled out with relatively strong expression of TP mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)


Culture-associated enhancement of LECAM-1 expression by lymphocytes and partial inhibition of enhancement by IL-4.

Recent studies have shown that the human leukocyte endothelial cell adhesion molecule-1 (LECAM-1) functions as a homing receptor, mediating leukocyte binding to high endothelial venules in peripheral lymph nodes. Increasing evidence has demonstrated that cytokines, such as IL-4, can modulate the expression of surface proteins such as homing receptors on a variety of cells. We thus investigated the modulatory effects of cytokines on LECAM-1 expression by lymphocytes using single- and dual-color flow cytometry. We found that the density of LECAM-1 expression increased markedly during 3 days of culture and that this culture-associated enhancement (CAE) of LECAM-1 expression was significantly inhibited by IL-4. B cells and both major T cell subsets (CD4, CD8) exhibited CAE of LECAM-1 expression, but the inhibitory effect of IL-4 on this response occurred only in the T cell populations. The inhibitory effect of IL-4 on enhanced LECAM-1 expression was reversible, and characterized all 3 LECAM-1 epitopes assessed. Natural killer cells, in contrast, did not exhibit CAE of LECAM-1 expression, and IL-4 had no modulatory effect on LECAM-1 expression by these cells. Another adhesion molecule, CD44, showed enhanced expression during culture, but this enhancement was not inhibited by IL-4. The results show that LECAM-1 expression by T and B lymphocytes is significantly increased during culture and that the inhibitory effect of IL-4 on this increase is restricted to T cells. These findings suggest that IL-4, generated during an immune response, may play a role in regulating the migration and localization of T lymphocytes to lymphoid tissues.


Aldosterone regulation of gene transcription leading to control of ion transport.

Aldosterone, like other steroid hormones, initiates its effects by binding to intracellular receptors; these receptors are then able to control the transcription of several genes. The products of these genes eventually modulate the activity of ionic transport systems located in the apical and the basolateral membrane of specialized epithelial cells, thereby modulating the excretion of Na+ and K+ ions. Considerable progress has been made recently in understanding these mechanisms and the structure of the proteins involved in these processes. A novel principle has been discovered to explain the selective effect of aldosterone on its target epithelia. These tissues exclude competing glucocorticoid hormones by the activity of the 11 beta-hydroxysteroid dehydrogenase to allow aldosterone, an enzyme-resistant steroid, to bind to its receptors. Aldosterone induces numerous changes in the activity of membrane ion transport systems and enzymes and cell morphology. Although the enhancement of Na,K-ATPase synthesis and the increase of the number of active Na+ channels in the apical membrane appear as both direct and primary effects, the mechanisms of the other effects remain to be determined. The knowledge of the primary structure of several elements of the aldosterone response system (e.g., mineralocorticoid receptor and Na,K-ATPase) allows us to understand abnormal regulation of Na+ balance at the molecular level and, potentially, to identify genetic alterations responsible for these defects.


Quantitation of hypothalamic atrial natriuretic peptide messenger RNA in hypertensive rats.

Previous studies from our laboratory have shown that spontaneously hypertensive rats have increased atrial natriuretic peptide stores and reduced norepinephrine release from nerve terminals in the anterior hypothalamus. We have postulated that atrial natriuretic peptide inhibits norepinephrine release in anterior hypothalamus, reducing excitation of sympathoinhibitory neurons, increasing sympathetic outflow, and elevating blood pressure in this model. The current study tested the hypothesis that atrial natriuretic peptide messenger RNA (mRNA) transcript levels are increased in anterior hypothalamus of spontaneously hypertensive rats compared with normotensive Wistar-Kyoto rats. Atrial natriuretic peptide mRNA in hypothalamic regions was measured by the quantitative polymerase chain reaction technique using a p-SELECT mutant atrial natriuretic peptide RNA as an internal standard. Atrial natriuretic peptide mRNA from hypothalamic regions of spontaneously hypertensive and Wistar-Kyoto rats and the internal standard were coamplified in a single reaction in which the same primers were used. Since the polymerase chain reaction product of the internal standard contained a new EcoRI restriction site, it could be distinguished from the atrial natriuretic peptide mRNA product by EcoRI digestion after the polymerase chain reaction. We found regional inhomogeneity of atrial natriuretic peptide mRNA in the hypothalamus of spontaneously hypertensive and Wistar-Kyoto rats, but we found no significant differences in atrial natriuretic peptide mRNA levels in anterior, posterior, or ventral hypothalamic areas between spontaneously hypertensive rats and Wistar-Kyoto rats fed normal (1%) or high (8%) salt diets. These data do not support the hypothesis that increased atrial natriuretic peptide stores in anterior hypothalamus of spontaneously hypertensive rats are related to increased gene transcription.


A synthetic glycoconjugate representing the genus-specific epitope of chlamydial lipopolysaccharide exhibits the same specificity as its natural counterpart.

The tetrasaccharide 3-deoxy-alpha-D-manno-2-octulosonic acid (alpha-KDO) (2----8)-alpha-KDO(2----4)-alpha-KDO(2----6)-beta GlcNAc, a partial structure of chlamydial lipopolysaccharide (LPS) representing a genus-specific epitope, was synthesized and covalently linked to bovine serum albumin, resulting in an artificial glycoconjugate antigen. Mice were immunized with the glycoconjugate to prepare chlamydia-specific monoclonal antibodies. They were selected with chlamydia-specific LPS antigens and the structurally and antigenically related Re-type LPS of a Salmonella minnesota rough mutant. Characterization of the selected antibodies was by (i) hemagglutination of sheep erythrocytes coated with recombinant chlamydia-specific LPS, (ii) inhibition by synthetic polyacrylamide derivatives containing the genus-specific epitope or partial structures thereof, (iii) enzyme immunoassay with recombinant LPS and synthetic bovine serum albumin glycoconjugates as solid-phase antigens, (iv) immunofluorescence of L929 monolayers infected with Chlamydia psittaci or C. trachomatis, and (v) Western immunoblots with glycoconjugates and LPS as the antigen. Two groups of monoclonal antibodies were obtained; the monoclonal antibodies in one group cross-reacted with chlamydial and Re-type LPS, but those of the other group were chlamydia specific. Among the latter, KDO trisaccharide-specific antibodies that had the same epitope specificity as antibodies obtained after immunization with chlamydial elementary bodies were identified; however, they exhibited a more than 100-fold higher affinity. In addition, antibodies that bound preferentially to the 2.8-linked KDO disaccharide were detected, although with lower affinity. The data show that the artificial glycoconjugate antigen is similar to its natural counterpart.


Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated.

The lipooligosaccharides (LOS) of strains of Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica contain epitopes that are antigenically and structurally similar to carbohydrates present in human glycosphingolipids. LOS from strains of Haemophilus influenzae and H. influenzae biogroup aegyptius were tested for the binding of monoclonal antibodies (MAbs) that bind to human glycosphingolipids possessing Gal beta 1-4GlcNAc (MAb 3F11) and Gal alpha 1-4Gal beta 1-4Glc (MAb anti-Pk). In solid-phase radioimmunoassays, the LOS of 18 of 19 H. influenzae type b (Hib), 8 of 19 nontypeable H. influenzae, and 10 of 20 H. influenzae biogroup aegyptius strains bound MAb anti-Pk. The LOS of 13 of 19 Hib, 10 of 16 nontypeable H. influenzae, and 2 of 18 H. influenzae biogroup aegyptius strains bound MAb 3F11. Neuraminidase treatment of the strains increased the binding of MAb 3F11 by more than twofold in 47% of the H. influenzae strains, suggesting that sialic acid occluded the LOS structure recognized by MAb 3F11. The material released from neuraminidase-treated Hib LOS was confirmed to be sialic acid by high-performance anion-exchange chromatography. A recombinant plasmid containing genes involved in Hib LOS biosynthesis directed the expression (assembly) of the 3F11 epitope in Escherichia coli. These studies demonstrate that H. influenzae and H. influenzae biogroup aegyptius express at least two LOS epitopes that are similar to those present in human glycosphingolipids. Sialic acid was present on the LOS of some H. influenzae strains and prevented the binding of MAb 3F11 to its epitope. The oligosaccharide portion of sialylated LOS may also resemble sialylated oligosaccharides present in human glycosphingolipids (gangliosides).


Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype.

Phosphatidylcholine, the major component of lung surfactant, when supplied as the sole source of phosphate for Pseudomonas aeruginosa PAO1, resulted in conversion of as much as 2% of the population to the mucoid phenotype under continuous culture conditions over a 24-day culture period. In addition, growth in phosphatidylcholine resulted in the highest yields of extracellular alginate compared with other environmental conditions. Iron limitation, another environmental condition relevant to the lungs of patients with cystic fibrosis, also resulted in conversion to mucoid. Since both conditions suggested the likelihood of an energy-deprived growth environment as a common variable, the effect of direct inhibition of energy generation by N,N'-dicyclohexylcarbodiimide or gramicidin on the conversion of nonmucoid P. aeruginosa to the mucoid phenotype was examined. Both inhibitors resulted in mucoid subpopulations (0.5 and 0.8%, respectively). Severe energy stress imposed by the combination of phosphate limitation and N,N'-dicyclohexylcarbodiimide treatment resulted in conversion of 55% of the population to mucoidy during a 7-day growth period. A growth advantage of the mucoid over the nonmucoid phenotype was observed under severe nutrient deprivation by growth on unsupplemented Noble agar or in a 1/2,500 dilution of a chemically defined medium. These results clearly demonstrate a significant role for the energy state of the cell in conversion to mucoid and in selection for the mucoid phenotype.


Stable, conserved outer membrane epitope of strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

Brazilian purpuric fever is a rapidly fatal childhood disease associated with a clonal strain of Haemophilus influenzae biogroup aegyptius. We describe a conserved, surface-exposed epitope present on 95% of H. influenzae biogroup aegyptius isolates that are associated with Brazilian purpuric fever. This epitope, defined by reaction with the monoclonal antibody 8G3, is on or associated with the 48-kDa heat-modifiable P1 protein. The epitope is absent on strains of H. influenzae biogroup aegyptius that are not associated with Brazilian purpuric fever but is present on one strain of H. influenzae biotype II. None of 81 other Haemophilus strains tested reacted with 8G3. The sensitivity and specificity of the 8G3 monoclonal antibody in detecting Brazilian case-clone strains of H. influenzae biogroup aegyptius associated with Brazilian purpuric fever are 95 and 99%, respectively. Immunoelectron microscopy revealed that the epitope is surface exposed, and N-terminal amino acid sequencing of an 8G3-reactive P1 protein from a strain of H. influenzae biogroup aegyptius showed 100% correlation with the published N-terminal amino acid sequence of a P1 protein of H. influenzae type b. The virulence of the organism in an infant rat model of bacteremia was not dependent on the expression of this epitope.


Isolation and characterization of recombinant antigens from Leishmania aethiopica that react with human antibodies.

A genomic expression library of Leishmania aethiopica was constructed in lambda gt11 and screened with patient sera and sera from healthy people living in an area of endemicity. Forty-five recombinant clones were isolated and partly characterized. Clone-specific antibodies were prepared and used with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis to estimate the molecular masses of the parasite-derived antigens containing the reactive epitope(s). Antigens with apparent molecular masses of 90, 85, 63, 50, 41, 25 and 24 kDa as well as several antigens with lower molecular masses were detected. The clone-specific antibodies from patients with diffuse cutaneous leishmaniasis reacted with high-molecular-weight antigens (30,000 less than Mr less than 90,000), whereas antibodies from patients with localized cutaneous leishmaniasis recognized low-molecular-weight antigens (Mr less than 25,000). Nine different purified recombinant antigens were obtained from lysogens in Escherichia coli Y1089 by immunoaffinity chromatography on anti-beta-galactosidase columns and were subsequently tested with patient sera. It is suggested that some of these recombinant antigens might be used for immunodiagnostic purposes.


Identification of the mucin-binding adhesin of Pseudomonas cepacia isolated from patients with cystic fibrosis.

In previous experiments, we have shown that isolates of Pseudomonas cepacia from sputa of patients with cystic fibrosis (CF), particularly those with severe lung infection, exhibited specific binding to purified respiratory or intestinal mucins (U. Sajjan, M. Corey, M. Karmali, and J. Forstner, J. Clin. Invest. 89:648-656, 1992). The present report describes the identification of the adhesin as a protein located on fimbriae of mucin-binding P. cepacia. From a total of 53 isolates available (from 22 patients with CF), we used three mucin-binding and three non-mucin-binding isolates for our experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P. cepacia homogenates was performed, the separated proteins were blotted onto nitrocellulose and overlaid with purified mucin, and mucin-binding components were detected with an antimucin antibody and then a second-antibody-alkaline phosphatase conjugate system. Only mucin-binding isolates exhibited a positively stained band at an Mr of 22,000. The 22-kDa protein was purified, and a polyclonal antibody specific for it was developed in rabbits. By electron microscopy and immunogold labelling, both the antibody and mucin (separately) were localized to pili present over the entire surface of the bacterial cells. Non-mucin-binding isolates did not have (or had very few) pili and did not stain with either mucin or the antibody to the 22-kDa protein. The purified 22-kDa protein and its antibody were each able to inhibit piliated P. cepacia binding to mucin. The amino acid composition of the 22-kDa protein was dissimilar to those of the major pilin proteins of Escherichia coli (type 1 pilus) and P. aeruginosa (PAK and PAO1 strains). Both the pili of P. aeruginosa PAK and PAO1 and antibodies to these pili failed to inhibit P. cepacia binding to mucin. Thus, P. cepacia adhesion to mucin is mediated by a pilin-associated 22-kDa protein which differs from epithelial-cell-binding pilin proteins of P. aeruginosa. We postulate that the 22-kDa adhesin may play a role in the virulence of P. cepacia lung infections of patients with CF.


Porphyromonas gingivalis fimbriae induce expression of the neutrophil chemotactic factor KC gene of mouse peritoneal macrophages: role of protein kinase C.

To account for infiltration of the periodontal tissues by neutrophils, the present study was undertaken to examine whether Porphyromonas gingivalis fimbriae, important structures involved in attachment of the bacteria to periodontal tissues, induce gene expression of the neutrophil chemoattractant KC in macrophages. The fimbriae induced expression of the KC gene of mouse peritoneal macrophages in a dose-dependent fashion. The peak of KC gene expression was observed as early as 1 h after initiation of the treatment. However, the gene expression was short lived, with the expression decreasing gradually after 6 h. A nuclear transcriptional assay showed that the fimbriae regulated the KC gene expression at a posttranscriptional level. We observed that the fimbria-induced KC gene expression was not regulated by endogenous or exogenous prostaglandin. Furthermore, forskolin, a potent activator of adenyl cyclase, and dibutyryl cyclic AMP were incapable of inducing KC gene expression of the peritoneal macrophages. H-8 and HA 1004, inhibitors of cyclic nucleotide-dependent protein kinases, had little effect on the fimbria-induced KC gene expression. On the other hand, the fimbria-induced KC gene expression was inhibited markedly by treatment with H-7, a potent inhibitor of protein kinase C. We also observed that phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, induced KC gene expression of peritoneal macrophages in a dose-dependent fashion. In addition, the fimbria-induced KC gene expression was suppressed in the peritoneal macrophages pretreated for 24 h with phorbol 12-myristate 13-acetate. These results suggest that the KC gene expression was mediated through activation of protein kinase C and not through that of cyclic nucleotide-dependent protein kinases. The present study indicates that P. gingivalis fimbriae can induce gene expression of the neutrophil chemotactic factor KC by macrophages via protein kinase C and suggests that this factor may be involved in infiltration of neutrophils into the periodontal tissues of adult periodontal patients.


Outer membrane protein patterns mark clones of Escherichia coli O2 and O78 strains that cause avian septicemia.

Major outer membrane proteins were isolated from 36 Escherichia coli strains representing six common clones of the O2 and O78 serogroups implicated in avian colisepticemia. Clonal relationships among isolates were inferred from an analysis of polymorphism at 20 enzyme-encoding loci detected by multilocus enzyme electrophoresis. For isolates of these clones, there was a high concordance (greater than 90%) between identity in multilocus genotype and major outer membrane protein patterns. The results indicate that major outer membrane protein patterns discriminate among the genetically different clonal groups that constitute the heterogeneous O2 and O78 serogroups associated with avian disease.


Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum.

The 47-kDa lipoprotein is an abundant integral membrane protein and dominant immunogen of Treponema pallidum subsp. pallidum. Previous DNA sequencing of the 47-kDa-lipoprotein gene did not reveal consensus features representative of other bacterial lipoprotein genes; this prompted further analyses with emphasis on elucidation of the N terminus of the molecule. To assist in localizing start signals for the protein, the transcription initiation site for the 47-kDa-antigen gene was determined. RNA isolated from both T. pallidum and recombinant Escherichia coli expressing the 47-kDa antigen was used as a template in reverse transcriptase primer extension. Upon analysis of cDNA products, transcription initiation was localized to one nucleotide in T. pallidum and to two adjacent nucleotides in E. coli. When various primers were used in DNA sequencing reactions for these analyses, a previously undetected nucleotide (G) was found in the purported 5' untranslated region; this altered the upstream reading frame to reveal plausible sites for ribosome binding (GGAGG), translation initiation (GTG start codon), and signal peptidase II processing (Val-Val-Gly-Cys). Discounting acylation, the molecular weight of the mature polypeptide is 45,756 (approximately 46,600 with acylation). To derive nonacylated 47-kDa antigen for further structure-function studies, the 47-kDa-antigen gene was subcloned without acylation signals as a genetic construct encoding a glutathione S-transferase fusion protein; following cleavage with thrombin, the nonacylated 47-kDa protein was hydrophilic rather than amphiphilic but retained its antigenicity when tested against 116 human serum samples from patients with various stages of syphilis.


Neutralization-sensitive epitopes are conserved among geographically diverse isolates of Cryptosporidium parvum.

Isolates of Cryptosporidium parvum from New York, Florida, Brazil, Mexico, and Peru were examined for the presence of two sporozoite surface epitopes originally identified in an Iowa isolate by neutralizing monoclonal antibodies (MAbs) 18.44 and 17.41. Immunofluorescence microscopy and immunoblotting demonstrated the presence of both epitopes on all isolates. Incubation of DEAE-cellulose-purified sporozoites of the New York, Florida, Brazil, and Mexico isolates with MAb 18.44 or 17.41 significantly neutralized their infectivity for 4- to 6-day-old BALB/c mice. The results indicate that two neutralization-sensitive epitopes are conserved on geographically diverse C. parvum isolates.


The circadian profile of extrasystolic arrhythmia: its relationship to heart rate and blood pressure.

This paper aims at examining whether there is an association between the circadian patterns of systolic blood pressure, heart rate and the incidence of ventricular ectopic beats, as well as to confirm that reducing the blood pressure by a diuretic may also reduce the ectopic frequency. Thirty-four ambulatory patients with ventricular ectopic beats and a systolic blood pressure of 131.33 +/- 17.46 mmHg had a 24-hour Holter electrocardiographic and blood pressure monitoring following 1 week off any antiarrhythmic and antihypertensive treatment. Then they received for one week a standard diuretic combination (amiloride 5 mg + hydrochlorothiazide 50 mg) at a dose depending on their systolic pressure value and their monitoring was repeated. The mean hourly values of systolic blood pressure, heart rate and ventricular ectopic beats were "normalized", i.e. expressed as (x-x)/SD, taking each patient's 24-hour average as zero and his own standard deviation as the unit of measurement. As a group, there was an independent positive correlation between blood pressure and ectopic beats, while the heart rate was a nonsignificant negative factor for ectopic beats. On an individual level, however, an independent positive significant correlation between blood pressure and ectopic beats was found in only 8 cases, with a negative one in 4 cases. While the blood pressure of the group ranged symmetrically around its daily average value, the corresponding ectopic beat curve was highly asymmetric, with a very high incidence (up to 2.56 +/- 0.52 SD) for a rather short time (only 9.41 +/- 3.56 hours above average) and a low incidence (up to 1.26 +/- 0.49 SD) for the remaining 14.59 hours below average. Sudden rises in ectopic beat (greater than 1 SD/hour) occurred 1 to 6 times per day in each individual, significantly (P less than 0.01) more often (20.31%) with a high (greater than 1 SD) blood pressure than with a low (less than -1 SD) one (8.99%) with intermediate frequencies at intermediate pressures. After treatment with the diuretic, the systolic blood pressure was reduced, the heart rate increased and the ventricular ectopic beat incidence reduced (significant changes). The mean change in systolic pressure in 25 patients with a reduction in ectopy was a significant (P less than 0.01) decrease (-5.21 +/- 8.70 mmHg) while in the remaining 9 cases there was a non significant increase (+1.68 +/- 7.63 mmHg). The heart rate was higher in both subgroups.(ABSTRACT TRUNCATED AT 400 WORDS)