A Ty1-copia group retrotransposon sequence in a vertebrate. We have used the polymerase chain reaction (PCR) to isolate a sequence characteristic of a Ty1-copia group retrotransposon from the genome of the herring (Clupea harengus). This is the first Ty1-copia group retrotransposon sequence described in a vertebrate. Phylogenetic comparison of this sequence with other members of this group of retrotransposons shows that it resembles more closely some Ty1-copia group members from Drosophila melanogaster than other group members in plants and fungi. These observations provide further evidence that the Ty1-copia group LTR retrotransposons span many of the major eukaryote species boundaries, suggesting that horizontal transmission between different species has played a role in the evolution of this retrotransposon group.


The suppressor of Hairy-wing binding region is required for gypsy mutagenesis. We undertook a deletional analysis of the gypsy retrotransposon in order to determine which sequences of the element are required for its mutagenic effect. We show that a phenotype indistinguishable from that of y2 flies can be generated by transforming y- flies with a construct containing the yellow gene and a gypsy element located at the same insertion site in yellow as found in y2 flies. When flies are transformed with similar constructs in which increasing amounts of the 5' transcribed untranslated region of gypsy have been removed, either a partial y2 revertant or a completely revertant phenotype is obtained. These results yield direct proof that the region of gypsy to which the su(Hw) protein binds is required for the generation of mutant phenotypes by this retrotransposon.


Retinoic acid receptors as regulators of human epidermal keratinocyte differentiation. To examine the role of nuclear retinoic acid (RA) receptors (RARs) in the regulation of squamous differentiation in normal human epidermal keratinocytes (NHEK), we analyzed binding activity, mRNA expression, and transcriptional activity of the endogenously expressed RARs. Specific RA-binding activity eluted from size-exclusion HPLC with an apparent mol wt of 50 kilodaltons and was predominantly (greater than 95%) associated with the NHEK nuclear cell fraction. This RAR-binding activity represented in part the expression of RAR alpha and RAR gamma genes, whose transcripts were expressed in similar abundance in undifferentiated NHEK. Differentiation resulted in lower mRNA expression of RAR alpha relative to the mRNA expression of RAR gamma. Treatment of NHEK cells with 10(-6) M RA did not induce expression of RAR beta mRNA. Similarly, three squamous cell carcinoma cell lines derived from human skin and oral cavity expressed RAR alpha and RAR gamma transcripts, but not RAR beta transcripts. Transfection of NHEK with chloramphenicol acetyltransferase (CAT) reporter plasmids indicated that the endogenously expressed RARs could activate transcription through the RAR beta response element in a concentration-dependent manner with doses of 10(-9) M RA and higher. CAT expression was not activated through TRE, a palindromic thyroid hormone response element with purported RA responsiveness. The competitive binding of benzoic acid derivatives of RA to RAR correlated with the ability of each analog to suppress mRNA expression of the squamous cell markers, involucrin, type I transglutaminase, and SQ37, and to activate transcription of the RAR beta response element-CAT reporter. These results demonstrate that the control of NHEK differentiation by RA is consistent with the interaction of the retinoid with RAR and the regulation of transcription by that ligand-receptor complex.


Immortalization of virus-free human placental cells that express tissue-specific functions. Human pregnancy-specific glycoproteins (PSGs) are a family of closely related placental proteins that, together with the carcinoembryonic antigen members, comprise a subfamily within the immunoglobulin superfamily. To facilitate study of the control of PSG expression, we immortalized human placental cell lines with adenovirus-origin-minus (ori-)-simian virus-40 (SV40) recombinant viruses containing either wild-type or temperature-sensitive (ts) A mutants of SV40. Cells transformed with the SV40 tsA chimera (HP-A1 and HP-A2), but not the SV40 wild-type chimera (HP-W1), were temperature sensitive for transformation. All three cell lines expressed trophoblast-specific genes, including PSG and the alpha- and beta-subunits of hCG. Human CG alpha expression was greatly stimulated by (Bu)2cAMP in all three cell lines; shifting HP-A1 and HP-A2 cells to the nonpermissive temperature (39.5 C) further increased hCG alpha expression. At both 33 C (permissive temperature) and 39.5 C, the transformed placental cells expressed PSG mRNAs of 2.2 and 1.7 kilobases; expression was greatly stimulated by sodium butyrate. In the absence of an inducer, the three placental lines synthesized a PSG of 64 kilodaltons (kDa). In the presence of butyrate, they synthesized PSGs of 72, 64, and 54 kDa, similar to the placental PSGs. However, in placenta the predominant species is the 72-kDa product. At 39.5 C, butyrate selectively increased synthesis of the 72-kDa PSG in HP-A1 and HP-A2 cells. To characterize PSG promoter activity, we constructed chloramphenicol acetyltransferase (CAT) fusion genes containing -809 to -44 basepairs up-stream of the translational start site of the PSG6 gene. Using transient expression assays, we demonstrated that the -809/-44 region of the PSG6 gene contained cis-acting sequences that can direct CAT expression in human placental cells. Sodium butyrate, which stimulates PSG expression, greatly increased CAT activity, indicating that butyrate-induced PSG expression is regulated primarily at the level of gene transcription.


Characterization of the 5'-flanking region of the rat thyrotropin receptor gene. Genomic clones containing 1.7 kilobases of the 5'-flanking region of the rat TSH receptor (TSHR) plus coding sequence from the ATG initiation codon [1 basepair (bp)] to the start of the first intron (170 bp) have been isolated and characterized. RNAase protection, primer extension, and cDNA sequences cloned by the anchored polymerase chain reaction identified multiple transcriptional start sites, the major ones clustered between -89 to -68 bp. This portion of the 5'-flanking region has neither a TATA nor a CCAAT box, is GC rich but has no GC box motif, and has features of promoters seen in "housekeeping" genes. Chimeras containing 1.7 kilobases (-1707 to -2 bp) of the 5'-flanking region, or deletions thereof, and the bacterial chloramphenicol acetyltransferase (CAT) gene expressed significant CAT activity when transfected into rat thyroid cell lines, FRTL-5 and FRT, but not BRL rat liver or HeLa cells. TSH decreased CAT activity in the FRTL-5 thyroid cells that had been stably transfected with the TSHR-CAT chimeric constructs. Negative regulation of promoter activity by TSH was duplicated by 10 microM forskolin in FRT thyroid cells, which express no TSHR mRNA. Deletion analyses indicated that a "minimal" region, exhibiting promoter activity, tissue specificity, and negative regulation by TSH, is located between -195 and -39 bp; this region is highly conserved in rat and human TSHR genes. Differential digestion of genomic DNA by MspI and HpaII revealed that the TSHR promoter is methylated in FRT, but not FRTL-5, cells; methylation of the promoter may be associated with loss of endogenous TSHR gene expression in FRT cells.


The unique C-termini of the thyroid hormone receptor variant, c-erbA alpha 2, and thyroid hormone receptor alpha 1 mediate different DNA-binding and heterodimerization properties. Thyroid hormone receptors (TRs) mediate the regulation of gene transcription by thyroid hormone (T3) by binding to T3-responsive elements (TREs) in target genes. c-erbA alpha 2 is a C-terminal TR variant which does not bind T3 and is a dominant inhibitor of T3 action. When synthesized in Escherichia Coli, alpha 2 formed two TRE-binding complexes similar to the monomeric and homodimeric forms of TR alpha 1. However, alpha 2 did not bind nearly as well as TR alpha 1. Furthermore, alpha 2 failed to bind DNA with proteins that heterodimerized with TR alpha 1. TR alpha 1 and alpha 2 also did not bind DNA as heterodimers with one another. The differences between TR alpha 1 and alpha 2 were further analyzed by studying a variety of C-terminal mutants synthesized in reticulocyte lysates. Deletion of the last 20 of the 122 unique amino acids (aa) of alpha 2 increased its DNA binding to approximately the level of TR alpha 1, indicating that the C-terminus of alpha 2 is an inhibitory domain. This alpha 2 mutant (alpha 2 delta C) was still unable to heterodimerize with nuclear proteins, as were C-terminal deletion mutants of TR alpha 1. We hypothesized that fusion of TR alpha 1-specific sequences to the C-terminus of alpha 2 delta C would transfer the property of heterodimerization. Indeed, although alpha 2/alpha 1 chimeras containing the last 40 and 70 aa of TR alpha 1 failed to heterodimerize with nuclear proteins, addition of the last 100 or 150 aa of TR alpha 1 did render alpha 2 delta C heterodimerization competent. Thus, TR alpha 1 contains a C-terminal structure which is necessary for heterodimerization and can confer this property on alpha 2, which lacks this domain. The effects of the unique C-termini of TR alpha 1 and alpha 2 on their in vitro DNA binding have important implications for their mechanisms of action in vivo.


Evidence suggesting an evolutionary relationship between transposable elements and immune system recombination sequences. Sequence similarity between the termini of invertebrate Tcl-like transposable sequences and the signal sequences of the vertebrate immunoglobulin somatic recombination pathway is described. These similarities suggest that the Tcl transposition pathway may share common sequence-specific binding factors with the immunoglobulin somatic recombination pathway.


Active specific immunotherapy with vaccinia melanoma oncolysate. Based on antitumor effects observed with vaccinia virus infected tumor cell lysate in animal models, adjuvant immunotherapeutic clinical trials were undertaken in patients with melanoma using vaccinia virus infected melanoma oncolysate (VMO). Preliminary clinical trials showed that the VMO is safe except minimal side effects such as mild fever, pain, and tenderness at the site of VMO injection, and mild lymphadenopathy. The effective dose of VMO was investigated in a following trial using 0.05 to 2.0 mg doses of VMO. Clinical responses and laboratory monitoring of melanoma-specific antibody responses decided the 2 mg dose of VMO is optimal for future trials. In all the clinical trials, patients showed moderate responses and their postimmune sera contained melanoma-specific antibodies. In a Phase II clinical trial completed August 1985 19 of 39 stage II patients had a disease-free mean survival time of 24.6 months, statistically significant compared with historical controls. Because of compelling evidence of significant clinical responses in patients treated with VMO adjuvant immunotherapy in the Phase II trial, a prospective randomized multi-institutional double-blind Phase III adjuvant VMO immunotherapeutic trial using adjuvant therapy of vaccinia virus alone as control, was recently initiated. Results of this trial are anxiously anticipated.


Delta and kappa opiate receptors in primary astroglial cultures. Part II: Receptor sets in cultures from various brain regions and interactions with beta-receptor activated cyclic AMP. In a previous paper, delta and kappa opiate receptors were shown to be co-localized on the same cell in enriched primary cultures of astroglia from neonatal rat cerebral cortex. Activation of the receptors inhibited adenylate cyclase. In this work, the presence of opiate receptors was investigated in astroglial primary cultures from neonatal rat striatum and brain stem. Cyclic adenosine 3',5'-monophosphate accumulation was quantified in the presence of different opioid receptor ligands after stimulation of the cyclic adenosine 3',5'-monophosphate system with forskolin. Morphine was used as a mu receptor agonist. [D-Ala2, D-Leu5]-enkephalin or [D-Pen2, D-Pen5]-enkephalin were used as delta receptor agonists and dynorphin 1-13 or U-50,488H were used as kappa receptor agonists. Specific antagonists for the respective receptors were used. After striatum or brain stem cultures had been incubated in 10(-9)-10(-5) M of each [D-Ala2, D-Leu5]-enkephalin, [D-Pen2, D-Pen5]-enkephalin and Dynorphin 1-13 or U-50,488H, dose related inhibitions of the 10(-5) M forskolin stimulated cyclic adenosine 3',5'-monophosphate accumulation were observed. The changes were reversed to the forskolin-induced control level in the presence of the respective antagonists. 10(-9)-10(-5) M morphine did not significantly change the forskolin-induced accumulation of cyclic adenosine 3',5'-monophosphate in the cultures studied. Furthermore, cultures from cerebral cortex, striatum or brain stem were incubated with isoproterenol alone or together with morphine or [D-Ala2, D-Leu5]-enkephalin.(ABSTRACT TRUNCATED AT 250 WORDS)


Heterozygosity for the "DN allele" (G533-greater than A) of the beta-hexosaminidase alpha subunit gene identified by direct DNA sequencing in a family with the B1 variant of GM2-gangliosidosis. A new patient having clinical, pathologic and biochemical features of the exceedingly rate B1 variant of GM2-gangliosidosis is described. This patient, of northern European (non-Ashkenazi) ancestry, is the first affected child of this ethnic background available for molecular genetic analysis; thus, she represented an opportunity to identify a new mutation associated with this phenotype or, conversely, to further characterize the DN allele of the hexosaminidase alpha gene (G533-greater than A) as a more widely distributed mutation not previously observed in this gene pool. As a means of rapid analysis, we report a strategy for PCR amplification and direct DNA sequencing of exon 5 of the hexosaminidase alpha gene, the most frequent site of mutations in this condition. With this technique, the father of the affected child was determined to be heterozygous for the DN allele, while the mother was found to have only the normal sequence in this region. This observation further extends the known geographic and ethnic distribution of this mutation, and suggests the likelihood that the DN allele has been derived by multiple independent mutational events.


Personality features and platelet 3H-imipramine binding. In an attempt to provide further information on the functions of the serotonergic system in humans, we investigated the possible correlation between a peripheral serotonergic marker, such as platelet 3H-imipramine binding, and the Minnesota Multiphasic Personality Inventory (MMPI) items and scales in a group of 20 healthy subjects. The results showed the presence of significant positive correlation between the maximum binding capacity, a measure of the number of 3H-imipramine sites, and 17 MMPI items suggestive of personality features of self-certainty, feeling of well-being and sense of reality.


Platelet alpha-2 adrenergic receptor binding and plasma free 3-methoxy-4-hydroxyphenylethylene glycol in depressed patients before and after treatment with mianserin. To examine the noradrenergic function in endogenous depression, binding of a selective agonist radioligand, 3H-UK14304, to platelet alpha 2-adrenergic receptors and plasma free 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) were measured in untreated depressed patients. The effects of an antidepressant, mianserin, on these parameters were also assessed. The Bmax and Kd values for 3H-UK14304 binding in 26 untreated depressed patients were significantly higher (p less than 0.05, p less than 0.01) than those in 26 normal controls. On the other hand, there were no significant differences in plasma free MHPG levels between 12 untreated depressed patients and 12 normal controls. Chronic administration of mianserin to 8 depressed patients slightly increased the Bmax and Kd values. However, plasma free MHPG levels did not change after treatment. These findings suggest that depression is related to the subsensitivity of alpha 2-receptors as indicated by a decreased affinity of platelet alpha 2-receptors. In addition, chronic administration of mianserin further decreased the affinity of alpha 2-receptors. This suggests that mianserin acts not only on alpha 2-receptors but also on the other neurotransmitter systems.


Placebo effects in standard human neuropharmacological studies: effects of physiological variations of blood glucose and ammonia concentration on the electrophysiology of the visual system. The effects on retinal and cortical visual evoked phenomena of the stimulus intensity/spatial frequency and of the spontaneous, physiological blood glucose/ammonia fluctuations were investigated comparatively in a pilot study on young male volunteers. Flash ERG and flash OPs to 5 different stimulus intensities and pattern VECP to 3 spatial frequencies were recorded at 2-hour intervals during a standard acute pharmaco-EEG experimental session (8 h) with administration of placebo; glucose and ammonia blood concentration levels were assessed concomitantly. The effects of the stimulus intensity/spatial frequency were statistically defined for each amplitude/latency measure by nonlinear regression analysis and were removed by computing the residuals from the regression function, which were then tested separately versus the glucose/ammonia concentration by linear regression. Glucose/ammonia statistically significant effects on the visual system were detected at concentration levels within the range of normality and were representative of a nonnegligeable portion of the data overall variance. These effects were selective on retinal/cortical evoked phenomena and it is conceivable that physiological or pathological metabolic changes might account for a still underestimated source of individual variability in human neuropharmacological studies otherwise adequately controlled.


Beta-endorphin and corticotropin immunoreactivity and specific binding in the neuromuscular system of obese-diabetic mice. Immunoreactivity for two derivatives of pro-opiomelanocortin, beta-endorphin and alpha-melanocortin (or corticotropin), was demonstrated, using a conventional immunoperoxidase method, in some of the intramuscular nerves in muscle sections from obese diabetic (ob/ob) mice and homozygous lean (+/+) mice. The endplate regions were visualized in the sections by staining for acetylcholinesterase reaction product. The proportion of muscle endplates with beta-endorphin-immunoreactive motor nerves was approximately 2.5-fold higher in soleus and extensor digitorum longus muscles and approximately 1.5-fold higher in the diaphragm of the obese (ob/ob) mice compared to the normal lean mice. The proportion of muscle endplates with alpha-melanotropin-immunoreactive motor nerves was between 30 and 53% lower, depending on the muscle type, in the ob/ob mice compared to the lean mice. The muscles of ob/ob and lean mice were investigated for the presence of specific binding sites for [125I]beta-endorphin and for [125I]corticotropin, using autoradiography. Some muscle fibres in soleus, extensor digitorum longus and diaphragm in both the ob/ob and the lean mice exhibited specific binding sites for the radioactive ligands. The binding sites were distributed over the entire surface in these muscle fibres. In the ob/ob mice the number of muscle fibres with specific [125I]beta-endorphin binding sites was six-fold higher in soleus and approximately 10-fold higher in extensor digitorum longus and diaphragm, than in the corresponding muscles of the lean mice. In contrast, the number of muscle fibres with specific [125I]corticotropin binding sites was similar in obese (ob/ob) and lean mice.(ABSTRACT TRUNCATED AT 250 WORDS)


Mediation of serotonin hyperalgesia by the cAMP second messenger system. In this study we have evaluated the second messenger system that might couple 5-HT1A receptor activation to produce peripheral hyperalgesia. The intradermal injection of the serotonin (5-hydroxytryptamine; 5-HT) receptor agonist for the 1A receptor subset (5-HT1A), (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthaline hydrobromide (8-OH DPAT) produces a dose-dependent hyperalgesia which was attenuated by a cAMP kinase inhibitor (the R-isomer of cyclic adenosine-3'-5'-monophosphate), but prolonged by the inhibition of endogenous phosphodiesterase by rolipram, supporting a role for the cAMP second messenger system. The 5-HT1A receptor agonist, 8-OH-DPAT, and the adenyl cyclase activator, forskolin administered together, produced an additive hyperalgesia, suggesting that the 5-HT1A receptor in peripheral terminals of the primary afferent neurons is positively coupled to the cAMP second messenger system in producing hyperalgesia. The inability of pertussis toxin to inhibit 8-OH DPAT-induced hyperalgesia further supports this hypothesis. The coupling of the 5-HT1A receptor to the cAMP second messenger system appears to be through guanine regulatory proteins since guanosine 5'-O-(3-thiotriphosphate) and cholera toxin both markedly enhanced 8-OH DPAT hyperalgesia. In further support of the role of guanine nucleotide regulatory proteins, guanosine 5'-O-(2-thiodiphosphate), as well as activators of inhibitory guanine regulatory proteins (the mu-opioid agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, and the adenosine A1 agonist, N6-cyclopentyladenosine, significantly attenuated 8-OH DPAT hyperalgesia.


Functionally distinct subpopulations of striatal neurons are differentially regulated by GABAergic and dopaminergic inputs--I. In vivo analysis. Two subpopulations of striatal neurons, Type I and Type II, are distinguished by their contrasting electrophysiological responses to paired impulse stimulation of cortical afferents. Although both Type I and Type II striatal neurons are excited by the first impulse of any pair of impulses, in response to short interstimulus intervals (10-30 ms) Type I neurons display an increase in probability of spike discharge to the second impulse (facilitation), whereas Type II neurons exhibit a decrease in probability of discharge (inhibition); in response to longer interstimulus intervals (50-250 ms) Type I cells display inhibition, whereas Type II cells show facilitation. The present experiments investigated the possibility that the unique paired impulse responses of Type I and Type II neurons reflect differential regulation by GABAergic and dopaminergic afferents. Extracellular recording techniques were combined with micropressure ejection of specific antagonists for GABAA (bicuculline), GABAB (phaclofen), D1 (R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-IH-3-benzazepin+ ++-7-ol; SCH23390) or D2 (sulpiride) receptors; the role of dopamine was also examined using the specific neurotoxin, 6-hydroxydopamine. Results showed that bicuculline (250-500 microM) reduced stimulation threshold for spike discharge of both Type I and Type II neurons and completely antagonized the paired impulse inhibition in response to short interstimulus intervals characteristic of Type II neurons. In contrast, phaclofen (2-30 mM) had only a variable influence on spike threshold for Type II cells and no effect on the paired impulse responses of either Type I or Type II neurons. Micropressure ejection of SCH23390 (1 mM) decreased spike thresholds for both cell types and attenuated the inhibition of spike discharge to long interstimulus intervals distinctive of Type I neurons, an effect which was mimicked by dopaminergic denervation. In contrast, sulpiride (1 mM) had little effect on spike thresholds, and no influence on the paired impulse responses of either cell type. These results indicate that the excitability of both Type I and Type II neurons is tonically inhibited by GABAergic and dopaminergic input via stimulation of GABAA and D1 receptors, respectively. Moreover, the bicuculline sensitivity of Type II neurons suggests that GABAergic input to this cell class arises from neurons within a cortically driven feedforward and/or feedback loop, whereas Type I cells receive input from neurons which lie outside of such a loop. In addition, the inhibition to longer interstimulus intervals characteristic of Type I cells is, at least in part, dependent on dopaminergic input through D1 receptor stimulation.


Serotonergic regulation of type II corticosteroid receptor binding in hippocampal cell cultures: evidence for the importance of serotonin-induced changes in cAMP levels. The development of the hypothalamic-pituitary-adrenal response to stress is profoundly altered by environmental events. One target for environmental regulation within the hypothalamic-pituitary-adrenal axis is the hippocampal type II corticosteroid (or glucocorticoid) receptor system, which mediates the negative-feedback effects of glucocorticoids on hypothalamic-pituitary-adrenal activity. Thus, adult rats handled early in life show increased hippocampal type II corticosteroid receptor density and increased sensitivity to the inhibitory effects of circulating glucocorticoids on post-stress hypothalamic-pituitary-adrenal activity. Both effects persist throughout life. The effects of handling on type II corticosteroid receptor development are, at least in part, mediated by changes in hippocampal 5-hydroxytryptamine turnover. Moreover, 5-hydroxytryptamine can regulate type II corticosteroid receptor density in cultured hippocampal cells, providing a paradigm for examining the neurochemical mechanisms by which environmental stimuli might regulate neural differentiation. In the present studies, we examined the intracellular mechanisms underlying the effects of 5-hydroxytryptamine on type II corticosteroid receptors ([3H]RU 28362 binding) in hippocampal cell cultures. cAMP, but not cGMP, levels in cultured hippocampal cells were significantly increased by the addition of 5-hydroxytryptamine to the medium. The cAMP response to 5-hydroxytryptamine was biphasic: an initial increase in cAMP levels occurred in response to nanomolar 5-hydroxytryptamine concentrations (EC50 = 7.2 nM), while a second increase was apparent at low micromolar concentrations. 5-Hydroxytryptamine also increased [3H]RU 28362 binding (EC50 = 4.5 nM) with a maximal effect at a concentration of 10 nM. There was no further increase in [3H]RU 28362 binding even with higher, micromolar concentrations of 5-hydroxytryptamine.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition by neosurugatoxin and omega-conotoxin of acetylcholine release and muscle and neuronal nicotinic receptors in mouse neuromuscular junction. Neosurugatoxin and omega-conotoxin, known to be specific ligands for the neuronal nicotinic receptor and Ca2+ channel, respectively, were previously claimed to exert no depressant action on the mouse neuromuscular junction. It was found that in preparations partially blocked with tubocurarine or with low Ca(2+)-high Mg2+ Tyrode's, both toxins, at 3-10 microM, depressed indirect twitches and either produced wanings (neosurugatoxin) or waxings (omega-conotoxin) of indirectly elicited tetanic contractions whilst in normal Tyrode's the contractile forces were not changed. In normal Tyrode's, neosurugatoxin decreased the amplitudes of spontaneous and evoked endplate potentials and enhanced the run-down of endplate potentials as did tubocurarine though with lesser potency. By contrast, omega-conotoxin (10 microM) decreased the amplitude of the evoked but not of the spontaneous endplate potential in low Ca(2+)-high Mg2+ Tyrode's, and produced facilitation of endplate potentials, instead of run-down, on repetitive stimulations. Higher concentrations of omega-conotoxin appeared to depress quantal release in normal Tyrode's. The effects were all reversible. The prolonged endplate depolarization found in preparations treated with neostigmine or 3,4-diaminopyridine, was partially depressed by both toxins. The results suggest that neosurugatoxin blocks the neuron and muscle nicotinic receptors in the neuromuscular junction with comparable potency. The pharmacology of the nicotinic receptor on motor nerve terminal seems more similar to the muscle nicotinic receptor than to that on autonomic ganglia or brain. On the other hand, omega-conotoxin seems to block a small fraction of Ca2+ channels on the motor nerve and decreases the quantal release of evoked endplate potentials.


Somatosensory evoked potentials in the evaluation of lumbosacral radiculopathy. We performed lower extremity somatosensory evoked potential (SEP) studies in 59 patients with signs or symptoms suggestive of lumbosacral radiculopathy and compared them with results of myelography with post-myelogram CT (myelogram/CT), MRI, and other electrodiagnostic studies. Of 38 patients with abnormal myelogram/CTs, 32 had abnormal SEPs, while 11 demonstrated EMG abnormalities. All 21 patients with normal myelogram/CTs had normal SEPs. SEP improved electrodiagnostic sensitivity in patients with weakness or reflex changes as well as in those with sensory deficits only. SEP was less sensitive in patients in whom spinal stenosis was the only radiographic finding. MRI generally corresponded well with the results of myelogram/CT and SEP but overestimated the significance of disk bulges in some patients. SEP is useful in the electrodiagnostic evaluation of lumbosacral radiculopathy, particularly when EMG is nondiagnostic.


Brain-adrenal axis hormones are altered in the CSF of infants with massive infantile spasms. Massive infantile spasms (MIS), a seizure disorder unique to infants, is considered an age-dependent response of the immature brain to various insults and stressors. The seizures improve with ACTH and glucocorticoids, both major components of the brain-adrenal axis. We hypothesized that CNS levels of these hormones are abnormal in infants with MIS and studied CSF from 14 infants with MIS and 13 age-matched controls by analysis for corticotropin-releasing hormone (CRH), ACTH, cortisol, and interleukin-1-beta. ACTH levels in CSF of patients were significantly lower than those of controls, but differences in cortisol levels between patients and controls were not statistically significant. CRH levels in both groups were similar and fluctuated diurnally. These results indicate an alteration of specific CNS components of the brain-adrenal axis in MIS.


No evidence for reduced thrombocyte cytochrome oxidase activity in Alzheimer's disease. We measured cytochrome oxidase activity in thrombocytes from patients with senile dementia of the Alzheimer type. We found no decrease in enzyme activity in Alzheimer's disease patients when compared with patients with multi-infarct dementia, with young control donors, and with age-matched control donors.


Needle-localized biopsy of occult breast lesions: an update. We have performed 207 needle-localized breast biopsies for nonpalpable, mammographically suspicious lesions over the past 6 years. A mass lesion and grouped microcalcifications were associated with malignancy, while a mass with microcalcifications was never associated with malignancy. Biopsy revealed malignancy in 26 cases (12.6%), with 19 of 22 (86.4%) having no histologic evidence of axillary spread. Advanced age and a past history of a breast cancer were again the risk factors present in a significant number of the patients with a positive biopsy result. There were 11 complications (5.3%): three hematomas (1.4%), one infection (0.5%), and seven (3.4%) required a repeat biopsy to remove the suspicious lesion missed on the original biopsy. We conclude that needle-localized breast biopsy continues to be a reliable method of detecting early breast carcinoma. Given the minimal morbidity, this procedure should be done in all patients with mammographically suspicious nonpalpable breast lesions.


A routine method for using sodium iodide to stabilize sodium pertechnetate [99Tcm] dispensed for the preparation of 99Tcm-exametazime. When preparing 99Tcm-exametazime, it is important to use sodium 99Tcm-pertechnetate that is less than 2 h old. The addition of sodium iodide (NaI) to 99Tcm-pertechnetate is known to extend this time to 6 h. This paper describes a technique for implementing this in routine practice. Sterile kits consisting of 440 micrograms NaI and 1.0 ml sodium chloride injection in a vial with a nitrogen atmosphere were prepared and stored at room temperature, 4 degrees C and -22 degrees C. Titrimetric analysis of iodide showed that under each storage condition, kits were stable for 8 weeks. To determine the effectiveness of the kits, the radiochemical purity (RCP) of 99Tcm-exametazime was measured by high-performance liquid chromatography (HPLC). The validity of this technique was determined by simultaneous analysis with the conventional thin-layer/paper chromatography (TLC/PC) technique on 24 occasions, over a range of RCP (94.5-54.6%). Radiochemical purities measured by HPLC and TLC/PC were 81.2 +/- 10.2 and 81.5 +/- 10.5%, respectively, and did not differ significantly (P greater than 0.30). The correlation between the techniques was high (r = 0.98). 99Tcm-exametazime was prepared using 1 h-old 99Tcm-pertechnetate, 6 h-old 99Tcm-pertechnetate and 6 h-old 99Tcm-pertechnetate dispensed in a NaI kit. At the recommended expiry time for this radiopharmaceutical, i.e. 30 min after preparation, RCPs were found to be 88.4 +/- 2.4, 80.9 +/- 2.0 and 89.3 +/- 3.0%, respectively (n = 5 for each technique).(ABSTRACT TRUNCATED AT 250 WORDS)


Monoclonal antigranulocyte antibody imaging in inflammatory bowel disease: a preliminary report. The 99Tcm-labelled antigranulocyte antibody BW250/183 has been used in the detection of intestinal inflammation in patients with active ulcerative colitis and Crohn's disease. Planar images were obtained up to 24 h after intravenous injection of the antibody. Eight out of nine patients with ulcerative colitis and six out of seven patients with Crohn's disease gave positive images. In 11 patients distribution of the inflammation was confirmed by barium studies, colonoscopy or surgery, whilst in two the antibody scan suggested more extensive disease than barium enema. None of the patients had any adverse reactions. Imaging with BW250/183 appears to give an accurate indication of the extent of inflammation in inflammatory bowel disease.


The prognosis and management of cervical cancer associated with pregnancy. We sought to evaluate the prognosis of patients with stage IB cervical cancer related to pregnancy. We reviewed 53 patients from the University of Michigan diagnosed with cervical cancer related to pregnancy between 1960-1989. Patients with stage IB disease were compared with a control group less than 46 years old to determine whether pregnancy influenced survival. The mean age of the patients was 30.5 years (range 22-45). The number of patients treated by 5-year periods decreased from a high of 17 during 1965-1969 to a low of three during 1985-1989. The following cell types and stages were treated: squamous cell stages IA (four), IB (35), and IIB (six); adenocarcinoma stages IB (five) and IIB (two); and small-cell stage IIB (one). The 35 patients with stage IB disease had a cumulative 5-year survival of 83%. Positive lymph nodes were present in eight of 24 patients. There was no significant difference in 5-year survival according to the time of therapy (P = .45): Ten second-trimester patients had 90% survival, five third-trimester patients had 75% survival, and 20 postpartum patients had 75% survival. Eighteen of 21 patients treated by radical hysterectomy survived: three of four treated at term, five of five treated in the second trimester, and ten of 12 treated postpartum. Seven of 12 patients treated by radiation therapy survived: one of one treated at term, five of six treated in the second trimester, and one of five treated postpartum. When we compared these patients with 170 nonpregnant women less than 46 years old with stage IB squamous cell cancer, pregnancy did not adversely influence survival (P = .13). The number of patients diagnosed with invasive cervical cancer related to pregnancy has decreased. Survival is not altered by pregnancy for stage IB disease.


Use of polyglactin 910 knitted mesh tubing to stabilize particulate hydroxyapatite in alveolar ridge augmentation. A preliminary report. A universal problem with the use of hydroxyapatite (HA) particles has been the tendency for these particles to migrate beyond planned boundaries. This is a preliminary report on the use of polyglactin 910 knitted mesh (Vicryl) tubing containing HA particles in ridge-augmentation procedures, assessing the technique and the containment of HA particles. The technique is described and experience with six cases is presented. This study found no clinical or radiographic evidence of particle migration after normal function had been resumed. In one case the graft failed to become firmly incorporated with the bone and the whole graft migrated anteriorly on the mandible but still provided improved function for the patient. The technique facilitates the placement of HA particles and would be particularly useful if open mucosal flap techniques were being used for ridge augmentation. The use of Vicryl tubing for HA augmentation procedures prevents the migration of particles and allows an easier and more accurate placement of the grafts.


Signet-ring cell adenocarcinoma metastatic to the maxillary sinus. Signet-ring cell variant is a rare type of adenocarcinoma that has been reported in the paranasal sinuses and in other areas, most commonly the gastrointestinal tract. We describe a patient with signet-ring cell adenocarcinoma of the maxillary sinus who had dental and facial pain. Further evaluation revealed that the lesion was metastatic from an esophageal primary lesion. The unusual nature of this cell type and the importance of careful evaluation to exclude the possibility of these lesions representing metastatic lesions is discussed.


Reaction of the dental pulp to hydroxyapatite. The purpose of this study was to evaluate the action of hydroxyapatite (HA) (Osteogen HA Resorb, GBD Marketing Group Inc., Valley Stream, N.Y.) on the dental pulp of rats. Four upper molar pulps in 45 rats were exposed and capped with synthetic HA (Osteogen) with a stereoscopic microscope. Pulps capped with calcium hydroxide (Dycal, L.D. Caulk Co., Milford, Del.) served as controls. The cavities were filled with amalgam, and the molars on each side of the maxilla were protected by the placement of a pedodontic steel crown. Pulp inflammation and dentin repair were compared by histologic observations and computer image analysis after 7, 14, and 28 days. After 7 days a partial acute pulpitis were observed in specimens treated with Osteogen or Dycal. Reparative dentin formation along the pulp walls was also seen. After 14 days the pulpitis was more extensive in the Osteogen-treated teeth than in the control teeth. Dentin formation as measured by morphometric analysis was more pronounced in Osteogen-treated teeth. Neo-odontoblasts were observed after the use of both materials. After 28 days an acute inflammatory reaction was still evident in the Osteogen-treated group. A complete dentinal bridge was observed more frequently with Dycal than with Osteogen. Despite the putative abilities of HA to be osteoconductive, osteogenic, and dentinogenic, the results of this study indicate that it should not be used as a pulp-capping agent because of its tendency to cause scattered dystrophic calcification in the dental pulp, which could interfere with future endodontic treatment.


[Veno-occlusive disease of the liver as a treatment complication in children with Wilm's tumor]. We report on three children aged 1 1/2, 2 and 9 1/2 years with Wilms' tumor, who developed a tender hepatomegaly and ascites associated with elevated liver enzymes, anemia and thrombocytopenia during chemotherapy. This clinical picture and liver sonography abnormality are best explained by veno-occlusive disease (VOD) of the liver, while other causes of liver disease could not be identified. Actinomycin D dosage was 0.045 mg/kg as bolus injection in two patients and 0.075 mg/kg split over five days in a third patient. Presumable, this drug was the causative agent. VOD was observed after preoperative and postoperative chemotherapy. No child had received abdominal irradiation. The authors comment on the influence of Actinomycin D as the cause for this unusual liver toxicity.


Erythrocyte sodium-potassium transport in cystic fibrosis. Erythrocytes from 15 patients with cystic fibrosis (CF) aged 8 mo to 22 y (mean age 12.8 y) were analyzed for Na+,K(+)-ATPase activity and sodium, potassium, and ATP concentrations. Sodium concentrations and Na(+)-K+ ratio of erythrocytes were statistically significantly lower in the CF patients [6.6 (SD 1.9) versus 9.2 (SD 1.1) mmol/L (p less than 0.01) and 0.070 (SD 0.023) versus 0.104 (SD 0.016) mmol/L (p less than 0.01), respectively]. The Na+,K(+)-ATPase activity was similar compared with that of reference individuals [536 (SD 100) versus 488 (SD 92) nmol inorganic phosphate/mg protein/h]. Intraerythrocyte sodium concentration and Na(+)-K+ ratio were thus lower in relation to the recorded Na+,K(+)-ATPase activities in controls, indicating a change of the passive transmembrane movements of sodium ions in CF. There was a rise of erythrocyte sodium and Na(+)-K+ ratio despite unchanged Na+,K(+)-ATPase activity after regular infusion of a fat emulsion rich in essential fatty acids, inferring that an altered membrane composition by essential fatty acid deficiency could explain the low intracellular sodium concentration in CF.


Expression of basic fibroblast growth factor and receptor: immunolocalization studies in developing rat fetal lung. To study the role of basic fibroblast growth factor (bFGF) in fetal lung development, the distribution of bFGF peptide and FGF receptor (FGF-R) was examined by immunohistochemistry in embryonic and fetal rat lung [d 12 to term (term = 22 d)]. Throughout development bFGF was localized to airway epithelial cells, their basement membranes, and their extracellular matrix. FGF-R was also detected in airway epithelial cells, especially in the branching areas, and in interstitial cells as early as d 13. The number of FGF-R immunoreactive cells increased during the embryonic and pseudoglandular stages of lung development, followed by fluctuations in reactivity during the canalicular stage. No FGF-R was detected in tissue from the saccular stage of lung development. The presence of bFGF and FGF-R in developing airway epithelium and mesenchyme is compatible with a role for this growth factor during fetal lung development. In the developing lung, bFGF seems to be sequestered and stored in the extracellular matrix, and may be released at times of need. Furthermore, FGF-R up- and down-regulation offers another mechanism by which the growth of specific cell populations may be controlled during fetal lung development.


Effects of thyroid hormone on myocardial adrenergic beta-receptor responsiveness and function during late gestation. To assess the effect of fetal hypothyroidism during late gestation on postnatal cardiovascular responsiveness, we measured heart rate and cardiac output responses to isoproterenol in newborn lambs. To evaluate the effect of such altered thyroid status on the development of beta-adrenergic signaling cascade, we measured myocardial beta-adrenergic receptor concentration and affinity, guanine nucleotide regulatory protein density, and adenylyl cyclase responsiveness. Twenty fetal lambs underwent either thyroidectomy and line placement or line placement alone at 128-130 d gestation. Five thyroidectomized and six control newborns were treated with isoproterenol, five thyroidectomized and four control newborns were killed upon delivery and tissue was obtained for biochemical studies, and four additional animals were delivered and killed at 126 d gestation and tissue was obtained for receptor analysis. Of the newborns treated with isoproterenol, the thyroidectomized lambs showed lower increase in heart rate and cardiac output compared with euthyroid newborns. Compared with the myocardium of normal newborns of similar gestation, the myocardium of the newborns who underwent fetal thyroidectomy failed to show the normal increase in beta-adrenoceptors accompanied by reduction in beta-adrenergic-stimulated adenylate cyclase activity. These results suggest that near term, the normal development of ovine fetus myocardial beta-adrenergic receptor is affected by thyroid hormones.


Developmental regulation of the sarcoplasmic reticulum calcium pump in the rabbit heart. Previous studies have demonstrated that myocardial function changes during mammalian perinatal development. The purpose of this study was to evaluate the subcellular basis underlying the slower relaxation in the developing heart by examining perinatal changes in sarcoplasmic reticulum (SR) function, and in SR Ca2+ pump protein and mRNA abundance. We measured Ca2+ uptake and ATPase rates in isolated fetal, newborn, and adult rabbit cardiac SR membranes. In fetal and adult SR membranes, we estimated the active Ca2+ pump protein content by measuring the steady state Ca(2+)-dependent phosphoenzyme content; the total Ca2+ pump protein content was estimated by Western analysis of the immunoreactive Ca2+ pumps. We isolated RNA from fetal and adult hearts and estimated the SR Ca2+ pump mRNA content by Northern analysis. Ca2+ uptake and ATPase rates were significantly lower in the fetal and newborn SR membranes compared with the adult. The contents of active and total Ca2+ pump protein and of Ca2+ pump mRNA were 52-63% lower in the fetus than in the adult. These results indicate that a great deal of the slower sarcoplasmic reticulum Ca2+ uptake and ATPase rates in the fetal rabbit heart can be related to lower Ca2+ pump mRNA and protein contents. It is evident that transcriptional and/or posttranscriptional regulation of the SR Ca2+ pump may form an important part of the subcellular basis of the perinatal change in mammalian cardiac relaxation.


Acute hemodynamic effects of converting enzyme inhibition in children with intracardiac shunts. The short-term hemodynamic effects of intravenous enalaprilat were assessed in 26 infants and children, aged 6 months to 15 years, with intracardiac shunts undergoing cardiac catheterization. Pulmonary and systemic pressure, flow, and resistance indices were measured by the direct Fick method before and 30 min after enalaprilat at 0.06 mg/kg. Aortic and pulmonary artery pressure decreased 15 and 20%, respectively, by 10 min, with little further change at 30 min. The heart rate did not change significantly and there was no reduction in systemic flow. In those with a large ventricular septal defect and normal or near-normal pulmonary resistance (less than 3.5 u.m2, n = 8), the mean pulmonary-systemic flow ratio decreased from 2.9 +/- 0.3 to 2.4 +/- 0.3 (p less than 0.05) and the mean left-to-right shunt from 7.4 +/- 0.8 to 5.9 +/- 0.7 L/min/m2 (p less than 0.02). Those with an elevated pulmonary vascular resistance (greater than 5 u.m2, n = 8) showed a varied response. Two children, both with Down's syndrome, an atrioventricular canal defect, and reversible pulmonary hypertension (as assessed by an infusion of isoproterenol), had no decrease in pulmonary vascular resistance with enalaprilat. There were no adverse effects. Converting enzyme inhibitors may benefit "heart failure" associated with large ventricular septal defects and normal or mildly elevated pulmonary resistance.


Focal dermal hypoplasia (Goltz syndrome): report of two cases with minor cutaneous and extracutaneous manifestations. Two women, ages 33 and 16 years, had focal dermal hypoplasia (Goltz syndrome) with unusual, minimal clinical manifestations. The lesions consisted of patchy, atrophic, scaly, telangiectatic macules arranged in a linear pattern along Blaschko's lines, involving the anterior and lateral aspects of both legs (patient 1) and the anterolateral aspect of the left leg (patient 2). Type I partial syndactyly involving the second and the third toes in both patients was also present. The clinical and histopathologic features and diagnostic difficulties of cases of this disorder with minimal cutaneous and extracutaneous manifestations are discussed.


Ki-1 large cell lymphoma with regressing lesions in a child. An 8-year-old boy was seen with a cutaneous Ki-1 anaplastic, large cell lymphoma with multiple lesions. Some of the lesions showed spontaneous regression. During more than seven years of disease no systemic involvement was observed, but recurrent, self-healing lesions did appear. Histopathologic examination of five lesions revealed a variety of findings, from an inflammatory infiltrate to a highly anaplastic pattern. The neoplastic cells expressed Ki-1 and leukocyte common antigens. Ultrastructurally, those cells showed ruffled indentations. The differential diagnosis includes microvillous malignant lymphoma. The patient has had a four-year follow-up without relapses.


A structural assessment of the apo[a] protein of human lipoprotein[a]. Apolipoprotein[a], the highly glycosylated, hydrophilic apoprotein of lipoprotein[a] (Lp[a]), is generally considered to be a multimeric homologue of plasminogen, and to exhibit atherogenic/thrombogenic properties. The cDNA-inferred amino acid sequence of apo[a] indicates that apo[a], like plasminogen and some zymogens, is composed of a kringle domain and a serine protease domain. To gain insight into possible positive functions of Lp[a], we have examined the apo[a] primary structure by comparing its sequence with those of other proteins involved in coagulation and fibrinolysis, and its secondary structure by using a combination of structure prediction algorithms. The kringle domain encompasses 11 distinct types of repeating units, 9 of which contain 114 residues. These units, called kringles, are similar but not identical to each other or to PGK4. Each apo[a] kringle type was compared with kringles which have been shown to bind lysine and fibrin, and with bovine prothrombin kringle 1. Apo[a] kringles are linked by serine/threonine- and proline-rich stretches similar to regions in immunoglobulins, adhesion molecules, glycoprotein Ib-alpha subunit, and kininogen. In comparing the protease domains of apo[a] and plasmin, apo[a] contains a region between positions 4470 and 4492 where 8 substitutions, 9 deletions, and 1 insertion are apparent. Our analysis suggests that apo[a] kringle-type 10 has a high probability of binding to lysine in the same way as PGK4. In the only human apo[a] polymorph sequenced to date, position 4308 is occupied by serine, whereas the homologous position in plasmin is occupied by arginine and is an important site for proteolytic cleavage and activation. An alternative site for the proteolytic activation of human apo[a] is proposed.


Proliferative effect of PGD2 on osteoblast-like cells; independent activation of pertussis toxin-sensitive GTP-binding protein from PGE2 or PGF2 alpha. PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cells. PGD2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 microM. The formation of inositol trisphosphate (IP3) induced by PGD2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGF2 alpha and both PGE2 and PGF2 alpha affected PGD2-induced IP3 formation additively. Pertussis toxin (PTX) inhibited both PGD2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 micrograms/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD2-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD2 was apparently different from that by PGE2 or PGF2 alpha. These results indicate that PGD2 activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2 alpha and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.


Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes. The synthesis and release of leukotriene B4 (LTB4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temperature and effects of calcium ionophore A23187 concentrations. Maximal LTB4 concentrations were determined when canine PMNs were incubated with 10 microM A23187. Increasing LTB4 concentrations were determined through 10 min incubation. The maximal LTB4 concentrations (310 +/- 30 pg LTB4/2.5 x 10(5) cells) determined at 10 min did not change through a 55 min incubation period. Greater LTB4 concentrations were synthesized by canine PMNs at 37 degrees C (268 +/- 12 pg LTB4/2.5 x 10(5) cells) than at 25 degrees C (206 +/- 11 pg LTB4/2.5 x 10(5) cells) or 5 degrees C (59 +/- 3 pg LTB4/2.5 x 10(5) cells). The synthesis of LTB4 in canine PMNs was inhibited by incubation of the cells with either of two known lipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB4 synthesis with an approximate IC50 = 0.1 microM, whereas BW755C inhibited LTB4 synthesis with an approximate IC50 = 10 microM. These results indicate canine PMNs have the capability to synthesize large quantities of LTB4 when stimulated with calcium ionophore A23187. Furthermore, the 5-lipoxygenase inhibitors BWA4C, an acetohydroxyamic acid, and BW755C, a phenyl pyrazoline, can readily inhibit LTB4 synthesis in canine PMNs.


Singlet oxygen induced DNA damage and mutagenicity in a single-stranded SV40-based shuttle vector. The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed. 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2. The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E. coli to study mutagenesis. There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA. It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.


Photosensitization by Photofrin II delivered to WI26VA4 SV40-transformed human fibroblasts by low density lipoproteins: inhibition of lipid synthesis and fatty acid uptake. Irradiation with 365 nm light of Wi26VA4 SV40-transformed human fibroblasts cultured for 24 h in the presence of low density lipoproteins loaded with the anticancer porphyrin mixture Photofrin II resulted in a near complete inhibition of [14C]oleic acid incorporation into triacylglycerols, cholesteryl esters and phospholipids. More than 80% reduction of the fatty acid incorporation in all lipid classes was observed following an irradiation dose of 1 J/cm2. The activities of the respective acyltransferases, measured in vitro on cell homogenates, were also markedly diminished, but to a lesser extent than lipid synthesis from oleic acid. Moreover, oleic acid uptake by cells was strongly and rapidly reduced. It is suggested that the rapid inhibition of membrane phospholipid synthesis upon cell photosensitization, due to both a direct inactivation of acyltransferases and to a reduction of fatty acid utilization, could play an important role in the photocytotoxic effect of Photofrin II.


Evaluating the drug dependency unit.

Evaluating the drug dependency unit. An evaluation of the St Mary's Drug Dependency Unit is described. Unit staff were interviewed and a retrospective analysis of 156 records was performed. The advent of HIV infection has led to an emphasis on public health priorities in the treatment of drug misuse. However, DDU staff retain a highly individualistic approach to their work. Staff aiming at abstinence within treatment may compromise important advice on harm minimization. The mean delay between first contact and entering treatment was 10 days; 42% of clients failed to complete the initial assessment. The need to extend access to the service is not easy to reconcile with the increasingly specialised support role for such units implied in recent strategy documents. Little is known of the effectiveness of different services for drug misusers. In this study, only 6% of clients were known to have achieved abstinence at one year. The efficiency of the DDU could be improved by closer definition of the unit's goals and target population, regular progress reviews, shortening the assessment procedure and the computerisation of data collection.


[Congenital mesoblastic nephroma in the adult (the first case of primary tumor in an adult over 40 years of age)]. Congenital mesoblastic nephroma CMN is an extremely rare renal tumor in adults, basicly seem in the childhood and, similar in some aspects to Wilms tumors. We present herein the first primary CMN in an adult older than 40 years and review the literature update. Three years after his operation (1991) the patient is clinically and echographically free of hepatic or abdominal recurrences. He has good renal function as determine by renogram, IVP and a normal serum creatinine.


[The dynamic magnetic resonance study of focal liver lesions by FLASH sequences with bolus intravenous gadolinium-DTPA]. Thirty-five patients with hepatic hemangioma (n = 12), metastasis (n = 10), hepatocellular carcinoma (HCC) (n = 10) and focal nodular hyperplasia (n = 3) were examined with the fast low-angle shot (FLASH) technique and an intravenous bolus injection of Gd-DTPA. In order to differentiate the lesions, the following criteria were used: a) pre Gd-DTPA intensity of lesions; b) post Gd-DTPA patterns of contrast enhancement. On the basis of these criteria, an unquestionable differential diagnosis could be made. Hemangiomas were characterized by an hypointense mass before Gd-DTPA, by peripheral contrast enhancement and by subsequent continuous hyperintense fill-in; thus, hemangiomas were visualized as hyperintense lesion during the late phase. Before contrast administration hypovascular metastases appeared as hypointense; they were characterized by delayed uptake of contrast agent. HCCs were hyperintense lesions before contrast administrations; then, quick contrast enhancement and rapid decrease in signal intensity were observed with visualization of a hyperintense ring due to the capsule. Finally, focal nodular hyperplasia appeared isointense or hypointense relative to normal liver on precontrast scans; the lesions were enhanced transiently with subsequent quick dismission of contrast agent. This initial experience suggests dynamic contrast-enhanced MR imaging as an effective method to improve the differential diagnosis among hepatic tumors when precontrast T2-weighted images are equivocal.


[The dynamic computed tomography of small hepatocarcinomas treated by US-guided percutaneous ethanol injections. The short- and long-term follow-up aspects]. Dynamic CT studies with an automatic injector of iodinated contrast medium were performed in 22 patients affected with 29 hepatocellular carcinomas (HCCs) (phi: 0.8-4.5 cm) before and after treatment with percutaneous ethanol injection (PEI) and during the follow-up period, every 6-9 months. Before PEI, most of the HCCs showed contrast enhancement on CT scans. Treatment was suspended when US-guided fine-needle biopsy demonstrated the absence of malignant cells and when the lesions were unenhanced on dynamic CT scans. Dynamic CT detected 21 HCCs (72.4%) before PEI and 24 HCCs (82.8%) after PEI and during the follow-up period. After PEI, 14 HCCs exhibited a thin and hyperdense peripheral rim on dynamic CT scans. Nine of these lesions, with long-term follow-up (12-31 months), have a smaller diameter than the primary lesion; 6 patients have no HCC recurrence. The authors conclude that dynamic CT is useful for evaluating the effectiveness of PEI in the treatment of HCCs; moreover, their personal experience suggests that the finding of a thin and hyperdense peripheral rim cannot always be related to viable cancerous tissue.


[The differential diagnostic criteria of breast microcalcifications]. The authors evaluated 127 cases (31 infiltrating carcinomas, 22 intraductal carcinomas, 74 benign lesions) of breast microcalcifications with no palpable lesions. The patients had undergone mammography, stereotaxic cytology and direct magnification (87 cases). Blind interpretation of standard films and of direct magnification was performed by two independent readers (A, B), who classified the cases according to radiologic pattern (annular, punctate, granular, linear, branching), degree of suspicion and need for surgical biopsy. The study confirmed the association of different types of microcalcifications with breast cancer (predictivity: annular, punctate, granular, linear, branching; A = 25%, 15%, 40%, 92%, 86%; B = 14%, 26%, 39%, 90%, 100%). However, diagnosis was not very accurate (sensitivity: A = 83%, B = 71.1%; specificity: A = 83%, B = 78.4%); it was also observed that most cancers can be diagnosed only at a relatively high cost in terms of unnecessary biopsies. Interobservers' agreement was high relative to diagnosis (negative/dubious vs. suspicious/positive = -88.9%; K = 58.5) and low for pattern attribution (52%). Direct magnification allowed better detail definition but if sensitivity improved (A = 87% vs. 80.6%; B = 64.5% vs. 51.6%), specificity was negatively affected (A = 73.2% vs. 80.4%; B = 69.6% vs. 80.3%), due to an excess of false positives. Stereotaxic cytology [inadequacy rate = 26%, sensitivity and specificity (ex inadequates) = 80% and 100%, respectively] was more accurate than mammography as far as the decision for surgical biopsy was concerned, and was correct in identifying most (A = 6/7, B = 7/11) of the cancers which had been misdiagnosed as benign at conventional and magnification mammography. Stereotaxic cytology should be routinely performed in cases of microcalcifications with no associated palpable lesions.


[The use of echography and US-guided percutaneous puncture in addition to mammography for the detection of malignant breast tumors]. At present, mammography is the most effective means to detect breast cancers, especially in the early stages. However, it lacks sensitivity and specificity in women with dense breasts. Moreover, indeterminate lesions are often seen on mammograms, which should undergo further examination before surgery. Due to recent improvement in the technique--i.e., the use of high-resolution 10-MHz transducers--US can now detect also nonpalpable breast lesions, about 1 cm phi. Fine-needle biopsy (FNB) under US guidance, which is complementary to US, allows a correct diagnosis of malignancy in a high number of cases. A total of 1821 women with indeterminate lesions at mammography underwent US, and 491 of them underwent US-guided FNB, in the Leno Hospital (Brescia, Italy), in the period 1988-90. Thirty-one breast cancers which had been missed at mammography and clinical examination were found. Three cases were carcinomas in situ, 23 invasive cancers were classified as pathological stage T1, and 15 cases had no axillary lymph node involvement. The routine use of US and FNB in addition to mammography when indeterminate lesions are seen on mammograms and in women with dense breasts may significantly reduce the number of both false-negative cases at mammography and unnecessary biopsies.


[Synovial sarcoma. A study of a series of 46 cases]. Synovial sarcoma is a rare malignant soft tissue tumor. The histological diagnosis remains sometimes difficult. The authors report 46 cases, treated in different hospitals in Paris. The medium survival rate of this group (which includes cases treated long ago) is of 30 per cent at 5 years. But only 24 per cent are disease free at this time. At the last follow up, the patients deceased after the fifth year included, the survival rate drops down to 12 per cent. Local recurrences and metastasis are analysed with reference to the different treatments applied. It seems that the association of polychimiotherapy and wide surgical excision might increase the survival rate, but this has to be confirmed in future. Radiotherapy is still a valuable treatment when excision is only limited, or when excision is impossible, due to localisation or extension of the tumor.


[Paralysis of the radial nerve and supracondylar fractures of the humerus in children. A study of a series of 11 cases]. Eleven radial nerve paralyses, associated with supracondylar fractures of the humerus in children, are reported: 2 incomplete motor, 5 complete motor, 2 complete motor and partial sensory and 2 complete motor and total sensory. The palsy was noticed before any treatment in 8 cases. In the 3 other cases, the palsy was secondary to difficult and repetitive closed reduction manoeuvers. The fractures were all of the extension type, grade III in 2 cases and IV in 9 cases. The displacement of the distal fragment was posterior and medial in 8 cases. Initial treatment of the fracture was conservative in 5 cases with 2 failures and surgical in 6 cases. Nerve function recovery always followed the same sequence: extensor muscles of the wrist, then extensor muscles of the fingers and at last, extensor muscles of the thumb. Time required for full recovery varied from 4 to 6 months. Open reduction and nerve control did not seem to be better than closed reduction with regard to the required time for neurological recovery and its quality. The indications depend on the extension of the paralysis. If radial palsy is incomplete, the first therapeutic step is closed reduction, followed by surgical reduction if not possible. A primary surgical approach is proposed in cases of complete sensory and motor paralysis because of the operative findings of contusion and incarceration of the nerve which may be aggravated by blind manoeuvers.


H2-receptors mediate histamine-induced variations in the permeability of the blood-brain barrier of rats. Earlier investigations suggested that histamine H2-receptors mediate alterations in cerebrovascular permeability in mammals. The objective of this study was to observe the effects of the H2-receptor antagonist ranitidine on histamine-stimulated alterations in the permeability of the blood-brain barrier (BBB) of rats. Normotensive Wistar-Kyoto (WKY) (control) and spontaneously hypertensive (SHR) rats were premedicated with ranitidine (1 mg/kg), followed by histamine (1.25, 2.5 or 5.0 micrograms/kg) or saline. Animals were anesthetized and permeability of the BBB was evaluated with 99mTc-labeled sodium pertechnetate (TcO4-) or with 131I-labeled human serum albumin (RISA). In both control and hypertensive rats, ranitidine blocked histamine-induced changes in cerebrovascular permeability, but did not inhibit histamine-stimulated systemic hypotension. The alterations in the permeability of the BBB did not correlate with variations in blood pressure produced by histamine.


[Segmental dystonia and mitochondrial encephalomyopathy]. A 55 year-old male experienced a dystonia of the right upper limb followed by a ptosis with complete ophthalmoplegia and cataract. He developed a sensory neuropathy and personality changes. Ragged-red fibers were found on muscle biopsy. There was a major defect in complex III and IV activity.


[Familial recurrent paralysis of the brachial plexus. Tomaculous neuropathy]. Familial recurrent plexopathy related to tomacular neuropathy is only the localized expression, with a familial predisposition, of a more diffuse disease. Such are the conclusions we drew from a study of 3 cases, in 2 families, of recurrent tomacular brachial plexopathy. In all 3 cases the condition was transmitted by the mother as an autosomal dominant trait. Biopsy of a sensory nerve was performed in 2 cases and showed, in one, typical images of myelinic degeneration at various stages, corresponding to the formation of tomacular tickenings. Tomacular brachial plexopathy must be distinguished from hereditary amyotrophic neuralgia.


[Monomelic ischemic neuropathy caused by subclavian artery obstruction]. A case study of acute ischemic monomelic neuropathy due to occlusion of the right subclavian artery is reported. The occlusion was embolic. Clinical examination showed sensory loss and severe distal weakness of the limb without obvious muscle necrosis. EMG showed motor and sensory nerve axon loss, chiefly distally. Despite surgical intervention, the neurological damage was irreversible.


[Causes and mechanisms of sciatic pains]. Pain along the sciatic nerve can be due to many causes. Sciatica of radicular origin caused by compression of L5 or S1 root is usually separated from truncular or peripheral sciatica. Radicular sciatica is divided into mechanical sciatica and the so-called inflammatory sciatica. In the majority of cases radicular pain is of mechanical origin and due to discal herniation in L4-L5 or L5-S1. The herniation can be visualized by lumbar computerized tomography or by radiculography. However, discal herniation alone does not fully account for the pain suffered, and inflammatory processes around the disc and the nerve root play an important role. Posterior intervertebral osteoarthritis and lumbar canal stenosis also are frequent causes of sciatic pain. Truncular sciatica is much less frequent and should incite clinicians to investigate for pelvic tumoral infiltration. Peripheral sciatica is usually caused by suffering of the external popliteal nerve around the neck of the fibula, but it may also be caused by compression of a ramus from the internal popliteal nerve, resulting in tarsal tunnel syndrome or in Morton's syndrome.


[Biology of the trophoblastic tissue and placental tumors]. The trophoblast of early placenta has many attributes of malignant tissue: it displays highly proliferative and invasive properties and expresses hormones, growth factors, growth factor receptors and oncogene products. Moreover, this tissue may have an autocrine control of growth. Collectively, these properties are similar to those of malignant tissues and the normal trophoblast is then considered as a "pseudomalignant" tissue. Moreover, either benign (hydatidiform mole) or malignant (choriocarcinoma) trophoblastic disease may be developed from the trophoblastic cells. In this article, the biological features of both the normal and the tumoral trophoblast will be presented. Finally, the trophoblast is a model and a source of molecules of biological interest.


[Gestational trophoblastic tumors].

[Gestational trophoblastic tumors]. The term gestational trophoblastic tumours covers hydatidiform moles, invasive moles and choriocarcinomas. These are rare diseases remarkable for their high chemosensitivity. The current objective is to cure all patients while preserving their potential for child bearing. Several studies have elicited prognostic factors, so that treatment can be adjusted to each individual patient. Advances in immunochemistry have led to the discovery of a highly reliable marker (HCG and beta-HCG) which has proved valuable to confirm the diagnosis and to follow the patients during and after treatment.


[3-day fever: agreement between clinical diagnosis and seroconversion of type 6 human herpesvirus (HHV-6)]. The aim of this study was to investigate the correlation between clinical diagnosis of exanthema subitum and seroconversion of herpes virus type 6 (HHV-6). 90 children with fever of unknown origin were investigated by 11 pediatricians in the Canton of Zürich, Switzerland, between October 1989 and June 1990. Antibodies against HHV-6 were determined at the first consultation and 2-3 weeks later. History and clinical findings were investigated and the clinical probability of the diagnosis exanthema subitum was estimated. 82 of the 90 children could be evaluated. 33 of them already had HHV-6 antibodies in their first blood sample although their history was negative concerning exanthema subitum. In 24 children exanthema subitum was proven by seroconversion. The clinical diagnosis was much more reliable at the second consultation compared to the first one. The clinical course of the disease was highly variable. Duration of fever differed widely and in some children the exanthema was atypical or even absent.


Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.


Distal esophageal obstruction due to a guar gum preparation (Cal-Ban 3000). Guar gum is a high polymer carbohydrate that undergoes considerable expansion and thickening when placed in water. Because of this property, guar gum has been used in "weight loss" tablets marketed by several pharmaceutical manufacturers. These tablets undergo expansion in the stomach and are expected to cause early satiety. Patients with esophageal disorders should be cautioned against the use of this product because it may expand in the esophagus and create an obstruction.


Morphology and laminar distribution of neuropeptide Y immunoreactive neurons in the human striate cortex. The morphology, laminar distribution, and distribution relative to cytochrome oxidase patches of neuropeptide-Y immunoreactive (NPY-ir) neurons were studied in the human striate cortex. The density of NPY-ir cells was highest in the white matter. NPY-ir neurons were sparsely distributed within the cortical layers. NPY-ir neurons were located in both cytochrome oxidase dense patch and interpatch regions. However, the paucity of NPY-ir neurons in layer III, where cytochrome oxidase patches are most clearly demonstrated, precluded establishing a clear relationship of NPY-ir neurons to cytochrome oxidase patches. NPY-ir neurons exhibited a variety of nonpyramidal morphologies, and many of them had axons with recurrent or looped trajectories. A dense plexus of NPY-ir axons was located just beneath the pia, and these axons were concentrated at the entry points of pial blood vessels. Other NPY-ir neurons had cell bodies or processes in close proximity to cerebral capillaries. These results suggest a role of NPY in cortical metabolism, control of cerebral circulation, or activity-related changes in local blood flow.


Dopamine depletion produces augmented behavioral responses to a mu-, but not a delta-opioid receptor agonist in the nucleus accumbens: lack of a role for receptor upregulation. Microinjection of either mu- or delta-opioid agonists into the nucleus accumbens produces an increased locomotor activity, and when the dopaminergic innervation of the nucleus accumbens is bilaterally lesioned, the locomotor response to the microinjection of mixed mu- and delta-opioid agonists is augmented. To determine whether the lesion-induced augmentation to opioids is specific to mu- or delta-opioid receptor activation, dopamine innervation of the nucleus accumbens was lesioned with 6-hydroxydopamine (6-OHDA), and the motor stimulant response to intra-accumbens microinjection of the selective mu-opioid agonist, Tyr-D-Ala-Gly-mePhe-Gly-OH (DAMGO), was compared to that of the delta-opioid agonist, [D-penicillamine2,5]-enkephalin (DPDPE). The lesions caused a 95% depletion of tissue dopamine levels in the nucleus accumbens of the DAMGO-injected rats compared to sham-lesioned rats. Horizontal and vertical photocell counts were significantly increased in response to DAMGO in 6-OHDA-lesioned compared to the sham-lesioned rats. This behavioral augmentation was dose dependent and blocked by naloxone. In rats with similar accumbal dopamine depletions (94%), the locomotor response to DPDPE was not enhanced. The augmentation in the behavioral response to DAMGO was not associated with a change in the Bmax or Kd of [125I]DAMGO binding in nucleus accumbens homogenates from lesioned rats. Likewise, using quantitative receptor autoradiography, no difference between 6-OHDA- and sham-lesioned rats was observed in [125I]DAMGO or [125I]DPDPE binding. Therefore, the augmented behavioral response to opioids in the nucleus accumbens following dopamine depletion relies predominately on mu-opioid receptor stimulation. However, this augmentation is not mediated by an alteration in the number or affinity of these receptors.


Membrane-bound and solubilized brain 5HT3 receptors: improved radioligand binding assays using bovine area postrema or rat cortex and the radioligands 3H-GR65630, 3H-BRL43694, and 3H-LY278584. Bovine area postrema tissue was used as a convenient source of tissue for studies of brain 5HT3 receptors. 5HT3 receptor density was determined to be 97 +/- 5 fmol/mg and 124 +/- 10 fmol/mg of protein using the commercially available 5HT3 radioligands, 3H-GR65630 and 3H-BRL43694, respectively. The equilibrium dissociation constants (KD) for 3H-GR65630 and 3H-BRL43694 were 0.5 +/- 0.1 nM and 1.7 +/- 0.3 nM, respectively. The affinities of a series of drugs for the 5HT3 receptor using the two radioligands were essentially identical; the Ki values and order of affinities of agonists and antagonists were very similar to data published in studies on radiolabeling of 5HT3 receptors in rat brain. 3H-LY278584 also labeled 5HT3 receptors in bovine area postrema homogenates with a KD of 3.1 +/- 0.1 nM and a Bmax of 84 +/- 6 fmol/mg. In rat cortical homogenates, 3H-LY278584 produced the most reliable specific signal of 72%, with a KD of 2.6 +/- 0.3 nM and a Bmax value of 10.5 +/- 1 fmol/mg. At 1 nM, 3H-GR65630 or 3H-BRL43694 specific binding represented 28 and 50% of total radioligand binding, respectively. These data in bovine and rat brain tissues indicate that bovine area postrema can be used with 3H-GR65630, 3H-BRL43694, or 3H-LY278584 for drug screening or molecular investigations of the brain 5HT3 receptor: only 3H-LY278584 can be used for studies on the regulation and/or the molecular properties of 5HT3 receptors in rat cortical homogenates. 5HT3 receptors were solubilized from bovine area postrema and characterized using 3H-GR65630.(ABSTRACT TRUNCATED AT 250 WORDS)


Coagulation inhibitors in pulmonary cancer patients. Pulmonary cancer patients are known to have an elevated risk to suffer from thromboembolic complications. Because hereditary deficiencies of coagulation inhibitors antithrombin III, protein C and protein S are known to cause thromboembolic events it was the aim of our study to search for acquired alterations of these proteins in pulmonary cancer patients. We could demonstrate antithrombin III and protein C to be within the normal range in patients suffering from pulmonary carcinoma. In contrast, in patients suffering from metastatic pulmonary carcinoma bound protein S was increased, while free protein S was significantly reduced. In some patients the decrease of free protein S was comparable to the diminution observed in hereditary protein S deficient patients. A high positive correlation was observed between C4b-binding protein and bound protein S, indicating C4b-binding protein to be a regulatory protein for the shift from free and anticoagulatory active to bound and anticoagulatory inactive protein S. In conclusion, the decrease of free protein S is one source for thromboembolic complications in pulmonary cancer patients. For interpretation of altered free protein S levels it is useful to measure C4b-binding protein.


Surveillance for CMV infection in orthotopic liver transplant recipients. Infection with cytomegalovirus is a major cause of morbidity and mortality following orthotopic liver transplantation. In order that preventive strategies may be devised, a detailed assessment of risk factors for infection and disease is required. We have prospectively assessed 46 orthotopic liver transplant recipients for CMV excretion from multiple sites in order to determine incidence of, and risk factors for, CMV infection and disease. Risk factors for posttransplant CMV infection were donor CMV seropositivity, an increased volume of peritransplant whole-blood transfusion and an increased dose of extra steroid therapy to treat rejection episodes. These findings implicate, respectively, transfer of virus with donor organ, transfer of virus with blood transfusion, and stimulation of reactivation of latent virus in recipients through augmented immunosuppression. The possible ways of preventing or ameliorating these effects are discussed.


Effect of repeated intradermal injections of bovine herpesvirus type 1 antigen on seronegative cattle. Forty-three cattle seronegative to bovine herpesvirus-1 (BHV-1) were given from one to five intradermal injections of BHV-1 inactivated antigen at four-week intervals. This delayed hypersensitivity test was assessed by the increase in skin thickness. The activity of the antigen was assessed in five animals which had a previous natural BHV-1 infection with clinical signs and seroconversion. Anti-BHV-1 antibodies were detected by seroneutralisation and an enzyme-linked immunoassay. Only one animal showed a significant but slight increase in skin thickness after the first test, but it was negative after a second test. The animals remained seronegative after the first test. Seroconversion was identified in 11 of the 43 animals (25 per cent) submitted to repeated delayed hypersensitivity tests. Five of 37 animals seroconverted after only two tests. The serological response was transient in seven of 11 seroconverted calves. Repeated hypersensitivity tests were therefore able to induce a serological response in seronegative calves but the response was weak and often transient. The test must therefore be applied cautiously to seronegative animals.


Suppression mediated in vitro by Marek's disease virus-transformed T-lymphoblastoid cell lines: effect on lymphoproliferation. Marek's disease virus (MDV)-transformed lymphoblastoid cells (MDCC-MSB1, -PA9 and -RP1) added to chicken splenic lymphocytes after treatment with mitomycin, suppress the lymphoproliferative response to T-cell mitogens (concanavalin A or phytohemagglutinin) by 40-70%. This suppressive activity was observed in syngeneic as well as in allogeneic combinations of cell lines and responder lymphocytes. The suppressive effect disappeared when the addition of MD-transformed cell lines to the responder cultures was delayed for 24 h. Treatment with glutaraldehyde, instead of mitomycin, greatly weakened the suppressive activity of the MD lymphoblastoid cells. A reduction of interleukin 2 (IL-2)-like activity produced by responder lymphocytes was observed after mixing with mitomycin-treated lymphoblastoid cells, but also, although slightly less, with the same glutaraldehyde-treated cells. Nevertheless no membrane fluorescence was observed, using INN-CH16 monoclonal antibody on MDV-induced lymphoblastoid cell lines to check up on the presence of IL-2 receptor-like structure. All the three lines exhibited a CD4+, CD8- phenotype.


The effect of dexamethasone-induced immunosuppression on the development of faecal antibody and recovery from and resistance to rotavirus infection. Rotavirus-naive and rotavirus-immune gnotobiotic calves were treated with high doses of dexamethasone (DX) to suppress the immune system. Calves were then infected with a virulent rotavirus inoculum, J-160, to investigate the role of immune responses both in recovery from primary rotavirus infection and in resistance to secondary rotavirus infection. Treatment of calves with DX markedly suppressed in vitro responsiveness of peripheral blood lymphocytes to mitogens within 48 h of the start of DX treatment. Suppression was similar in rotavirus-naive and rotavirus-immune calves. In contrast, the effect of DX treatment on specific antibody responses differed depending on when DX treatment started in relation to rotavirus infection. When DX treatment commenced prior to primary rotavirus infection both systemic and local specific antibody responses were inhibited. These calves, in which mitogen and antibody responses were suppressed, exhibited greater clinical signs than did control calves after infection with virulent rotavirus, but virus excretion was affected in only one of the two calves. When DX treatment was started after primary rotavirus infection but before secondary infection, systemic and local antibody responses to the primary infection and to the challenge infection were not affected. These calves resisted challenge with virulent virus as did DX-untreated rotavirus-immune calves, even though mitogen responses were suppressed. We conclude that in a primary rotavirus infection, virus excretion ceased when both antibody and mitogen responses were suppressed. Resistance to secondary rotavirus infection occurred when mitogen responsiveness was suppressed, but when antibody levels were normal. Thus, no evidence was obtained that fully functional cell-mediated immune mechanisms are essential for resistance to rotavirus infection. Evidence was provided for the ability of parenteral treatment with DX to suppress mucosal as well as systemic antibody responses.


Three-dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryo-electron microscopy. When the viral proteins VP3 and VP7 of bluetongue virus (BTV) are expressed simultaneously in the baculovirus system, core-like particles form spontaneously. The 3-D structure of these core-like particles, determined from cryo-electron micrographs, reveals an icosahedral structure 72.5 nm in diameter with 200 triangular spikes arranged on a T = 13,I lattice; The five spikes around each of the fivefold axes are absent. This is in contrast to the native BTV core particles which have a complete T = 13,I lattice of 260 spikes. The spikes, attributed to VP7 trimers appear as triangular columns 8.0 nm in height with distinct inner and outer domains. The inner shell of the core-like particles, or subcore-like particle, has a T = 1 lattice composed of 60 copies of VP3. The subcore-like particle is noticeably thicker around the fivefold positions. Pores in the subcore-like particle are situated near each of the local sixfold axes, below each six-membered ring of spikes. These pores could allow the passage of metabolites and RNA to and from the core for RNA transcription during infection. It is possible that the synthetic core-like particles have an incomplete complement of VP7 spikes because the ratio of VP7 to VP3 produced in the dual expression system is less than the 13:1 required for complete core-like particles. Only the VP7 spikes which have the strongest affinity for the VP3 inner core and are involved in maintaining the structural integrity of the core-like particle are incorporated. The BTV core-like particle shows greater morphological similarity to the rotavirus than to the reovirus core particle.


Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization. We have analyzed human papillomavirus (HPV) type 16 RNA expression in premalignant cervical lesions of different severity and in squamous cervical cancers by RNA-RNA in situ hybridization in order to find differences in the topographic distribution of viral RNA, which might correlate with the severity of disease. In the basal layer of low-grade squamous intraepithelial lesions (SIL) only weak transcription of viral early genes was observed. Signal intensity increased strongly in the more differentiated cells accompanied by high levels of HPV DNA replication. This pattern of viral gene expression, together with the onset of viral late transcription in the upper differentiated layer of the epithelium, most likely reflects the productive phase of viral infection. In contrast, in high-grade SIL viral transcription was comparatively strong in basal cells and evenly distributed throughout the undifferentiated epithelium. This difference of viral transcription in the basal layer of the respective lesions points to an altered regulation of viral gene expression which may be causally linked to the progression of precursor lesions. Evidence for disrupted expression of 3' early genes (E2, E4, and E5), analogous to the situation in HPV-DNA containing cervical carcinoma-derived cell lines, was not found in any of the HPV-16-positive premalignant lesions nor in the majority of cancers. The similarity of the viral transcription pattern of high-grade SIL and cancers suggests that additional host gene alterations are necessary for malignant progression.


Targeted gene disruption in Epstein-Barr virus. We report the development of a method that should allow the insertion of a selective marker into any region of the Epstein-Barr virus (EBV) genome of strain B95-8 through homologous recombination with plasmids. In this method, EBV recombinants are isolated as G418-resistant, immortalized B-cell clones or as G418-resistant, latently infected subclones of Burkitt lymphoma cell lines. The presence of the productive replication origin of EBV, oriLyt, on the plasmid was found to increase the number of observed recombinant viruses by approximately 100-fold; this stimulation was observed when oriLyt was separated from the sites of recombination by several kilobases of nonhomologous DNA. Long segments of EBV DNA flanking the marker on the plasmid and/or a large plasmid size were inferred to be important for obtaining a high proportion of recombinant genomes that had recombined on both sides of the selective marker; otherwise, the recombinants that predominated had acquired the entire plasmid by recombining only on one side of the inserted marker. Therefore, to facilitate targeted insertion of genetic markers into the EBV genome, a cosmid vector carrying oriLyt was constructed and tested by using it to generate EBV mutants with the BALF2 open-reading frame disrupted.


Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events. Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of greater than 320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.


Analysis of the bovine viral diarrhea virus genome for possible cellular insertions. Mucosal disease is the most severe disease resulting from bovine viral diarrhea virus (BVDV) infection in cattle. Two biotypes of BVDV may be isolated from animals with mucosal disease: cytopathic (cp) and noncytopathic (ncp). These "pairs" of cp/ncp viruses are often closely related and it has been suggested that the cp virus arises from a ncp virus by insertion of cellular RNA in the p125 region of the BVDV genome. We have used four pairs of cp/ncp BVDV isolated from cattle with mucosal disease, to examine the genomic sequence of the region of the genome coding for the nonstructural protein p125 (processed to p54/p80 in cp viruses) by PCR analysis and sequencing. We did not detect any cellular gene insertions in any of the four ncp viruses; however, we found a large duplication of the p80 gene and a ubiquitin gene insertion in three of the four cp isolates. Our results suggest that cellular RNA insertions in the p125 region may contribute significantly to the cytopathogenicity of BVDV. However, this does not appear to be the only mechanism of cytopathogenicity as we did not detect any insertions or duplications in one of the cp viruses. Comparison of the DNA sequence in the p80 region revealed greater homology within the "pairs" than to NADL, which lend further support to the hypothesis that a cp virus is originated from a ncp virus.


The DNA sequence of equine herpesvirus-1. The complete DNA sequence was determined of a pathogenic British isolate of equine herpesvirus-1, a respiratory virus which can cause abortion and neurological disease. The genome is 150,223 bp in size, has a base composition of 56.7% G + C, and contains 80 open reading frames likely to encode protein. Since four open reading frames are duplicated in the major inverted repeat, two are probably expressed as a spliced mRNA, and one may contain an internal transcriptional promoter, the genome is considered to contain 76 distinct genes. The genes are arranged collinearly with those in the genomes of the two previously sequenced alphaherpesviruses, varicella-zoster virus, and herpes simplex virus type-1, and comparisons of predicted amino acid sequences allowed the functions of many equine herpesvirus 1 proteins to be assigned.


The pathogenicity of Ab4p, the sequenced strain of equine herpesvirus-1, in specific pathogen-free foals. The sequencing of the genome of equine herpesvirus-1 (EHV-1) is reported in Elizabeth A. R. Telford, Moira S. Watson, Kathryn McBride, and Andrew J. Davison, 1992, Virology, 189, 304-316. The sequence was derived using a plaque-purified clone of EHV-1 strain Ab4 (designated Ab4p). To ensure that Ab4p shares the pathogenic characteristics of parental Ab4 (hereafter Ab4), both were inoculated intranasally into foals, specifically free from EHV-1 and EHV-4. Clinical signs, including rectal temperature, were similar for both viruses. In addition, nasal shedding of virus was observed over a 1- to 2-week period postinfection, and viremia was established with both Ab4 and Ab4p. Isolation of virus from one foal following intravenous administration of steroids indicates that Ab4p can establish latency and undergo reactivation. Finally, retinal lesions were observed and these were similar to those seen with Ab4. In conclusion, several pathogenic characteristics of Ab4 are retained in the plaque-purified clone, Ab4p.


Inhibition of bovine papillomavirus plasmid DNA replication by adeno-associated virus. The helper-dependent human parvovirus adeno-associated virus type 2 (AAV) inhibits both the oncogenic transforming abilities and the DNA replication of its helper viruses, adenovirus (Ad), and herpes simplex virus (HSV). As AAV-2 also inhibits the transforming ability of bovine papillomavirus type 1 (BPV), AAV-2 was assayed for its ability to inhibit BPV plasmid DNA replication. Here we find that the AAV-2 Rep78 gene is able to trans-inhibit BPV plasmid DNA replication and that the AAV-2 terminal repeats (TR) are also cis-required for the full inhibitory effect of AAV-2. When both the AAV-2 Rep78 open reading frame and TRs are present the inhibition of BPV plasmid DNA replication is very strong.


Lytic infection of primary rhesus kidney cells by simian virus 40. In an attempt to analyze the persistent infection of rhesus monkey cells with Simian virus 40 (SV40) in vitro, as described previously (reviewed in L. C. Norkin, Microbiol. Rev. 46, 384-425, 1982), we infected primary rhesus cell cultures (PRK), derived from a SV40-free monkey colony with SV40. Surprisingly, SV40 infected PRK cell cultures released as much infectious virus as cultures of the permissive African green monkey kidney cell line TC7. Infected PRK cells exhibited typical symptoms of a lytic infection, and the bulk of infected PRK cells died within 8 days postinfection (p.i.). A considerable proportion of infected PRK cells exhibited distinct SV40-caused cytopathic effects (CPE), similar to CPE in infected TC7 cells. We conclude that the in vivo persistence of SV40 in rhesus monkeys is not determined by cellular host factors, but by the immune system of the infected animals.