[Effects of consanguineous marriages on hereditary diseases: a study of the Han ethnic group in different geographic districts of Zhejiang Province]. The frequency and effects of hereditary diseases of consanguineous marriage (CM) in 210 965 Han nationality people in different districts of Zhejiang province were investigated. Of 44342 couples in three different regions, 951 (2.14%) were proved to be of CM. The frequency of CM was 3.02% in Mi as against 1.13% in Pi and 2.50% in Ii. There were 19 types and 81.07% of CM occurred in the first cousins, of which 0.95% occurred in semisiblings. The frequency of hereditary disease and the mortality before the age of 20 in the offsprings of CM were significantly higher than those of non-CM (P < 0.01). 104 types of hereditary disease and congenital malformation were found. The frequency of mental retardation was 2/1000. There were 197 high risk families which contained 19-types of hereditary disease.


[Choice of ultrasound fetal weight estimation formulae]. Ultrasound estimation of fetal weight has been widely used in obstetrics practice and become an assistant diagnostic method for intrauterine growth retardation (IUGR) and large gestational age (LGA). To assess the accuracy of different fetal weight estimation formulae, 1300 fetuses with gestational age ranging from 28 to 40 weeks, whose mothers had no pregnant complications, were estimated by using four selected formulae (R2 > 95%). The results were compared with the average birth weight at the same gestational weeks in 15 cities of China. 188 fetuses were subjected to ultrasound examination one week before their birth. The estimated weights by different formulae were compared with their birth weights. 46 IUGR fetuses and 57 LGA fetuses, who were delivered in our hospital, were used to evaluate the diagnostic accuracy of the four fetal weight estimation formulae. The ideal formulae and the way to improve the accuracy of the fetal weight estimation were discussed.


[One-stage extralong neurovascular pedicled segmental muscle flap transfer in the treatment of late facial paralysis]. One-stage extralong neurovascular pedicled segmental muscle flap transfer was used in the treatment of late facial paralysis in 10 cases. The length of ultralong neurovascular pedicle (14-17.5 cm) was longer than that of normal muscle flap (3.5-8 cm), since the former included dorsal thoracic artery, vein and nerve with their branches and ending segments as a whole. Compared with the current operations, the design of this operation can omit the cross-facial nerve graft in the first stage of operation and prevent the possible failure of additional nerve anastomosis. Ten cases were followed up for more than a year. All of them revealed voluntary movement of the transferred muscle flap with satisfactory results.


[Production of monoclonal antibodies against Mycobacterium leprae]. A series of hybridoma cell lines, which secrete monoclonal antibodies (McAbs), were produced by means of fusion between mouse myeloma cells SP2/O and spleen cells from BALB/C mice immunized with whole M. leprae plus unique phenolic glycolipid I(PGL-I) of M. leprae and M. leprae sonicates supernatant fluid (MLSS) as immunogen. Primary identification indicated that H2 cell line can secrete McAb against the epitope of PGL-I; IIIE10 cell line can secrete McAb against PGL-I and MLSS and (5) 24 D6C8 cell line only against whole M. leprae. The uses of these McAbs in serodiagnosis of leprosy, identification of M. leprae, analysis and purification of M. leprae antigens, and key problems in technology for producing McAbs against M. leprae were also discussed.


[A highly sensitive diagnostic kit for evaluating therapeutic effect in schistosomiasis cases]. A highly sensitive diagnostic kit for detecting circulating antigens of schistosomiasis was developed. It can detect the circulating antigen of this infection at a level of 10(-9) g/ml for the purposes of diagnosis and treatment. In acute cases of schistosomiasis, the detecting rate was 100% in 45 cases, and 93.0%-100% in 621 cases of early chronic stage with various infection loads. False positive rate was 0.1% in 513 normal individuals and 438 cases of other parasitic infections. The results showed that the kit was of high sensitivity and specificity. The variation coefficients in single batch and between batches were found to be less than 10%, indicating its good stability and reproducibility. Serum samples from cases treated by praziquantel were tested with the kit with a negative reaction rate of 79.7% 3 months after treatment. The kit can be used for diagnostic and therapeutic purposes.


An AF-DX 116 sensitive inhibitory mechanism modulates nicotinic and muscarinic transmission in cat superior cervical ganglion in the presence of anticholinesterase. The effect of the muscarinic receptor antagonist AF-DX 116 on the inhibitory action of muscarinic agonists and on responses mediated by nicotinic or muscarinic ganglionic transmission was studied in the superior cervical ganglion of the anesthetized cat. The postganglionic compound action potential evoked by cervical sympathetic trunk stimulation was depressed by methacholine or acetylcholine (ACh) injected into the ganglionic arterial supply. The depression was blocked by AF-DX 116. The compound action potentials evoked by preganglionic stimulus trains were also depressed when the intratrain frequency was 2 Hz or greater. This intratrain depression was, however, insensitive to AF-DX 116. The anticholinesterase drug physostigmine markedly enhanced the intratrain depression of the compound action potential. This effect was reversed by AF-DX 116. During nicotinic receptor block with hexamethonium, preganglionic stimulus trains with intratrain frequencies of 5 Hz or greater produced nicitating membrane contractions that could be blocked by the M1 muscarinic receptor antagonist pirenzepine. The amplitude of the contractions increased with frequency and reached a maximum at 20-40 Hz. AF-DX 116 had no effect on these responses. After administration of physostigmine, the amplitude of the nictitating membrane responses decreased with increasing intratrain frequency. AF-DX 116 reversed this effect. The data suggest that, in the superior cervical ganglion, AF-DX 116 sensitive muscarinic receptors which depress synaptic transmission are activated by exogenous agonists but not by the ACh released by the preganglionic axon terminals unless cholinesterase activity is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)


The pituitary gonadotropin-releasing hormone (GnRH) receptor of the female rabbit: characterization and developmental aspects. The aim of the present study was to characterize the pituitary gonadotropin-releasing hormone (GnRH) binding site in the rabbit and investigate its possible role in sexual maturation of the female rabbit. A radioligand binding assay was established, and the presence of specific 125I-labelled D-Ala6-des-Gly10-GnRH ethylamide (125I-DAl6EA) binding sites in the anterior pituitary gland of the rabbit was demonstrated. 125I-DAla6EA binding was saturable, specific, displaceable, reversible, correlated with increasing tissue concentrations, and susceptible to physiological manipulation. 125I-DAla6EA binding indicated the presence of two binding sites in the female adult rabbit pituitary: a high affinity, low capacity site (KD = 0.3-0.4 nM; Bmax = 100-200 fmol/mg protein) and a lower affinity, high capacity site (KD = 30 nM; Bmax = 5-8000 fmol/mg protein). Ontogeny of 125I-DAl6EA binding in the female rabbit (40-120 days of age) did not show a correlation between binding site number and serum luteinizing hormone (LH). In addition, the net serum LH response in female rabbits to a subcutaneous injection of DAla6EA (10 ng, 100 ng, and 1 microgram per kilogram body weight) was not significantly different between animals 40, 75, and 120 days of age. This suggests that a decrease in pituitary responsiveness to GnRH is not associated with sexual maturation in the female rabbit. Results indicate that factors other than and (or) in addition to GnRH binding site number, such as postreceptor events, play a role in gonadotropin secretion in the female rabbit.


[Rapid detection of anti-HAV IgM by solid-phase immunosorbent hemagglutination inhibition test]. A solid-phase immunosorbent hemagglutination inhibition test (SPISHAI) was developed for hepatitis A virus-specific immunoglobulin M (IgM) antibody. Three hundred thirty and six sera were comparatively detected with both SPISHAI and ELISA. Among them 97 sera were positive and 237 were negative with both method. The crude agreement rate was 99.4%. With SPISHAI the titers of anti-HAV IgM ranged from 1:20 to 1:327,680 among tested sera from infected individuals by HAV. The specificity of SPISHAI was confirmed by 2-ME treatment method and blocking test. The patients with non-A hepatitis all got negative results. The SPISHAI does not require conjugated antibody and sophisticated equipment, and is not interfered with rheumatoid factor in sera. Furthermore, the result of the test can be got within 3 hours. Therefore, the SPISHAI is a cheap and simple, and could be applied for early diagnosis and epidemiological surveillance of hepatitis A in the community and in primary health care.


[Surgical treatment of carcinoma of the middle and lower rectum. Report of 949 cases]. Carcinoma of the rectum in 949 cases (484 cases of middle rectum and 465 cases of lower rectum) was resected from 1954 to 1986. There were 296 (31.99%) cases of sphincter-saving resection (SSR). The results of SSR before 1980 were compared with those after 1980. The operative mortality of curative resection decreased from 1.52% to 0.34%. The overall survival rate was 74.20 +/- 2.16% (life table method), for abdominoperineal resection (A-P resection) was 61.67 +/- 3.62% and for SSR was 84.12 +/- 2.40% Respectively. The local recurrence after curative resection in all cases was 14.02%, after A-P resection-15.44%, and after SSR-10.92%. The local recurrence after SSR before 1980 was just the same as after 1980. The results in this series showed the rationality of SSR in the treatment of carcinoma of the middle and lower rectum.


Differential action of (-)-baclofen on the primary afferent depolarization produced by segmental and descending inputs. The purpose of the present series of experiments was to analyze, in anesthetized and paralyzed cats, the effects of (-)-baclofen and picrotoxin on the primary afferent depolarization (PAD) generated in single Ib afferent fibers by either intraspinal microstimulation or stimulation of the segmental and descending pathways. PAD was estimated by recording dorsal root potentials and by measuring the changes in the intraspinal activation threshold of single Ib muscle afferent fibers. The PAD elicited by stimulation of group I muscle or cutaneous afferents was readily depressed and often abolished 20-40 min after the intravenous injection of 1-2 mg/kg (-)-baclofen. In contrast, the same amounts of (-)-baclofen produced a relatively small depression of the PAD elicited by stimulation of the brainstem reticular formation (RF). The monosynaptic PAD produced in single Ib fibers by intraspinal microstimulation within the intermediate nucleus was depressed and sometimes abolished following the i.v. injections of 1-2 mg/kg (-)-baclofen. Twenty to forty minutes after the i.v. injection of picrotoxin (0.5-1 mg/kg), there was a strong depression of the PAD elicited by stimulation of muscle and cutaneous afferents as well as of the PAD produced by stimulation of the RF and the PAD produced by intraspinal microstimulation. The results obtained suggest that, in addition to its action on primary afferents, (-)-baclofen may depress impulse activity and/or transmitter release in a population of last-order GABAergic interneurons that mediate the PAD of Ib fibers. The existence of GABAb autoreceptors in last-order interneurons mediating the PAD may function as a self-limiting mechanism controlling the synaptic efficacy of these interneurons.


Further evidence for myelinated as well as unmyelinated fibre damage in a rat model of neuropathic pain. A mononeuropathy, produced by ligation of the sciatic nerve in rats, has recently been proposed as an animal model of experimental pain and pain-related disorders (hyperalgesia and allodynia). We investigated quantitatively the morphological changes in myelinated and unmyelinated fibres of the sciatic nerves 2 weeks after ligation in rats exhibiting allodynia to thermal stimulation. There was a marked reduction in the number of large myelinated fibres distal to the ligature (711 +/- 34 compared with 5315 +/- 230 in normal nerves). We also found a significant loss of small myelinated fibres (2429 +/- 109 compared with 3197 +/- 308 in normal nerves), the remaining fibres of this type showing pathological properties. Finally, ultrastructural evidence of damage to unmyelinated fibres was found. The typical pattern of large clusters of normal unmyelinated axons was no longer present within most regions of the nerve. There was a significant reduction in the size of the unmyelinated fibres (0.41 micron +/- 0.15 compared with 0.71 micron +/- 0.08 in normal nerves), together with a twofold increase in their number per cluster. Hypotheses about the mechanism of thermal allodynia in this pain model therefore must take into account the fact that all fibre classes show pathological changes.


Paradoxical signal transduction mechanism of endothelins and sarafotoxins in cultured pituitary cells: stimulation of phosphoinositide turnover and inhibition of prolactin release. Endothelins (ET-1, ET-2, ET-3 and vasoactive intestinal contractor, VIC) and sarafotoxins (SRTX-b and SRTX-c) appear to bind with high affinity to a homogeneous class of binding sites in cultured rat pituitary cells. All of these ligands seem to interact with the same receptor (ETA-R), except for SRTX-c which apparently binds to a separate receptor. Binding was followed by phosphodiesteric cleavage of phosphoinositides, resulting in the formation of inositol phosphates. No consistent effect on basal or gonadotropin-releasing hormone (GnRH)-induced release of luteinizing hormone (LH) was exerted by ET or SRTX during 2 h of static incubation. On the other hand, both groups of vasoactive peptides inhibited basal and thyrotropin-releasing hormone (TRH)-induced prolactin secretion. Surprisingly, activation of phosphoinositide turnover by TRH in pituitary mammotrophs led to stimulation of prolactin secretion, whereas activation of the same pathway by ET or SRTX resulted in inhibition of prolactin secretion. ET and SRTX stimulated inositol phosphate formation in GH3 cell line and in the gonadotroph-like cell line alpha T-3 (which is capable of producing the alpha subunit of the gonadotrophins), indicating that the peptides interact with both pituitary mammotrophs and gonadotrophs. The very low concentrations (nM range) needed to stimulate phosphoinositide turnover and to inhibit prolactin secretion, as well as the recent finding that ETs are present in the hypothalamo-pituitary axis suggest that ET might participate in the neuroendocrine modulation of pituitary functions. One such possibility is that ETs might be members of the prolactin inhibiting factors (PIFs) family.


Assessment of the role of cyclic nucleotides in allatostatin-induced inhibition of juvenile hormone biosynthesis in Diploptera punctata. In an effort to identify the signal transduction mechanism associated with the inhibition of juvenile hormone (JH) biosynthesis by the neuropeptides allatostatins, levels of the cyclic nucleotides cAMP and cGMP were measured in corpora allata (CA) of virgin and mated Diploptera punctata females using radioimmunoassays. Treatment of isolated CA with varying concentrations of synthetic allatostatins 1, 2, 3 or 4 did not elicit significant changes in the levels of either cAMP or cGMP in any of the test glands, suggesting that these compounds do not act as second messengers for the four allatostatins tested. Simultaneous treatment of CA with allatostatin 4 and the adenylate cyclase activator forskolin did not increase the degree of inhibition of juvenile hormone biosynthesis relative to that obtained with forskolin (5 or 50 microM) alone. We interpret these results as lending further support to the suggestion that cyclic nucleotides do not play a role in the signal transduction of allatostatins 1-4 in cockroach CA.


Effects of pituitary hormones on the cell-specific expression of the KAP gene. The expression of kidney androgen-regulated protein (KAP) gene in mouse kidney is regulated in a multihormonal fashion. As determined by in situ hybridization analysis, epithelial cells of proximal convoluted tubules of cortical nephrons express KAP mRNA in response to androgenic stimulation while similar cells in the juxtamedullary S3 segment of the tubules express KAP mRNA under estrogenic and pituitary hormonal control. In situ hybridization analysis of kidney sections using hypophysectomized (hypox) mice resulted in a total absence of KAP mRNA suggesting the participation of a pituitary hormone(s) in the constitutive expression of KAP mRNA in S3 cells. Treatment of hypox mice with steroid hormones showed that androgens restored the ability of cortical tubule cells to synthesize KAP mRNA. Estrogen treatment, on the other hand, partially induced KAP gene expression only in S3 cells. These results indicated that the androgenic response of the gene is independent of pituitary function, while expression in S3 cells, although partially induced by the direct action of estrogens, is primarily regulated by a pituitary factor. In order to elucidate which hormone(s) is responsible for KAP gene expression in S3 cells, individual pituitary hormones were administered to hypox normal animals and to strains of mice genetically deficient in certain pituitary hormones. Surgically treated C57BL/6 female and male mice were implanted for 7 days with osmotic pumps containing individual pituitary hormones, after which the kidneys were analyzed by in situ hybridization. Mice injected with growth hormone (GH), corticotropin (ACTH), prolactin (PRL), or vehicle failed to express KAP mRNA. Mice treated with thyrotropin (TSH), follitropin (FSH), and lutropin (LH) exhibited high levels of KAP mRNA in S3 cells of females as well as in the renal cortex of male animals. Expression in the cortex in response to LH and FSH may be due to their gonadotropic effect on testosterone production. Similarly, contamination of TSH samples with small amounts of the gonadotropins may explain the cortical response to TSH. TSH produced the strongest response in S3 cells suggesting that it is responsible for the permissive effect of the pituitary on KAP gene expression. This conclusion was supported by studies performed with the dwarf mouse (dw/dw) which lacks PRL, GH, and TSH due to a mutation in the pit-1 gene. In situ hybridization analysis of dwarf mice kidney sections showed a complete lack of KAP gene expression. The possible participation of GH and PRL was eliminated on the basis of the hormone replacement studies.(ABSTRACT TRUNCATED AT 400 WORDS)


Basic fibroblast growth factor enhances testosterone secretion in cultured porcine Leydig cells: site(s) of action. The effect and mechanism of action of basic fibroblast growth factor (bFGF) on testicular steroidogenesis were investigated using as a model primary cultures of purified porcine Leydig cells from immature intact animals. Basic FGF increased basal and human chorionic gonadotrophin (hCG)-induced testosterone accumulation (with an ED50 of 0.64 ng/ml bFGF, 35 pM) in the medium following a long-term treatment. The effects of bFGF (10 ng/ml, 72 h) were found at all hCG concentrations tested (0.001-1 ng/ml), the growth factor affecting the maximal steroidogenic capacity of the Leydig cells but not their sensitivity to the gonadotrophin. In this context, we have therefore investigated whether the stimulatory effect of bFGF on testosterone formation was related to an increase of the steroidogenic enzyme activities. The data obtained indicate that the growth factor did not affect the gonadotrophin action on the formation of delta 5-steroid hormone, namely dehydroepiandrosterone (DHEA) (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase). By contrast, bFGF (10 ng/ml, 72 h) was found to increase in a comparable manner the conversion of pregnenolone, DHEA and delta 4-androstenedione into testosterone, suggesting a stimulatory effect on 17 beta-hydroxysteroid dehydrogenase activity. Indeed, bFGF enhanced in a dose-dependent manner (ED50 = 39 pM) this enzyme activity evaluated through the conversion of delta 4-androstenedione to testosterone. These effects of bFGF on Leydig cell steroidogenic activity are probably exerted through specific membrane bFGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching. Cultured bovine adrenocortical cells reach replicative senescence after 100-120 population doublings in culture. Before reaching senescence, cells undergo high frequency phenotypic switching from CYP17+ to CYP17-, where '+' and '-' refer to the ability of intracellular cyclic AMP to induce expression of CYP17 (steroid 17 alpha-hydroxylase). We used luciferase reporter constructs to assess the activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching. We constructed two plasmids containing -2544 to +29 and -488 to +29 of the 5' region of CYP17 linked to a promoterless luciferase gene. Because of technical difficulties with transient transfection of late-passage bovine adrenocortical cells, these experiments were performed using stable transfection. Cells at early passage (PDL 10) and late passage (PDL 55) were cotransfected with either of these two plasmids ligated to pSV3neo, and G418-resistant pools of clones were derived. The activity of the CYP17 promoter in these transfectants was tested by growing cells in complete medium until semiconfluent and then transferring them into defined medium with cholera toxin and insulin-like growth factor I for 6 h. Luciferase activity was consistently induced by cholera toxin/IGF-I over five passages in pooled clones derived by transfection of early passage cells with the -488 construct. Despite the lack of expression of the endogenous CYP17 gene in transfectants from late-passage cells, induced luciferase activity was higher in late-passage transfectants than early-passage transfectants for both the -2544 and -488 constructs.(ABSTRACT TRUNCATED AT 250 WORDS)


Binding of sex-hormone-binding globulin (SHBG) to testicular membranes and solubilized receptors. Sex-hormone-binding globulin (SHBG) binds to a specific protein on the surface of prostate, epididymis, and a human breast cancer cell line (MCF-7), and is internalized by these cells. The present study demonstrated specific binding of SHBG to receptor on membranes prepared from rat testes. The binding was saturable, specific, and time and temperature dependent. Scatchard analysis of these binding studies suggested that SHBG binds to a single class of sites on testicular membranes with a Kd at 37 degrees C of 5 x 10(-8) M and a binding capacity of 30 +/- 0.6 pmol/mg protein. These binding characteristics are similar to the SHBG receptor on human prostate and MCF-7 cells. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (158 +/- 0.3 pmol/mg protein) and a 10-fold decrease in binding affinity (Kd at 37 degrees C = 6.5 x 10(-7) M). The apparent molecular weight of the testicular SHBG receptor, as estimated by gel filtration, was M(r) = 174,000. a specific binding site for SHBG was identified on testicular membranes. This binding site has been tentatively identified as a SHBG receptor based on its physical properties in testicular membrane preparations and following solubilization.


Molecular cloning of the bovine prolactin receptor and distribution of prolactin and growth hormone receptor transcripts in fetal and utero-placental tissues. We have isolated a bovine prolactin (bPRL) receptor cDNA from an endometrial cDNA library, which predicts a 557 amino acid transmembrane protein similar to the long forms of other characterized prolactin receptors. The predicted cytoplasmic domain is slightly truncated primarily by a stop codon located 36 codons 5' from the stop utilized in the human hepatic transcript. When expressed in COS cells, this cDNA was shown to encode a protein which bound bovine placental lactogen (bPL) and bPRL with nearly equal affinity (KD for bPL, 2.03 x 10(-10) M; bPRL, 3.07 x 10(-10) M). Northern analysis demonstrated multiple transcripts, with maternal liver, corpus luteum, intestine, endometrium and fetal liver containing a major transcript of about 3.8 kb, and maternal corpus luteum and endometrium, a second sized transcript of apparently equal abundance of 4.4 kb. This difference did not appear to be within the coding region. Primer extension analysis of maternal hepatic and endometrial transcripts revealed considerable heterogeneity. Examination of the distribution of prolactin and growth hormone receptor transcripts at mid-pregnancy by semi-quantitative reverse transcriptase polymerase chain reaction showed that both are widespread in bovine fetal and placental tissues. This isolation of bovine prolactin receptor cDNA, and description of receptor distribution will facilitate study of the action of the placental and pituitary members of this gene family during pregnancy.


Aldosterone-specific membrane receptors and rapid non-genomic actions of mineralocorticoids. Functional studies in extrarenal, non-epithelial cells such as smooth muscle cells and more recently circulating human lymphocytes have provided increasing evidence that aldosterone produces not only classical genomic effects, but also rapid, non-genomic effects on transmembrane electrolyte movements. These involve activation of the sodium/proton exchanger of the cell membrane at very low, physiological concentrations of aldosterone with an acute onset within 1-2 min. A second messenger cascade involved is the inositol 1,4,5-trisphosphate/calcium pathway which responds over the same rapid time course. Such changes clearly cannot be explained by genomic mechanisms, which are responsible for later effects than the membrane related rapid responses. The mechanisms underlying these rapid effects of aldosterone on electrolytes have been extensively studied in human lymphocytes, which thus may represent valuable tools in the delineation of the receptor-effector mechanisms involved. The unique characteristics of this new pathway for steroid action include its rapid time course, 10,000-fold selectivity for aldosterone over cortisol and the ineffectiveness of spironolactones, classical mineralocorticoid antagonists, as antagonists of the response.


Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. We have isolated the gonadotropin-releasing hormone receptor (GnRH-R) from a rat anterior pituitary cDNA library, determined its sequence and demonstrated receptor function. The 2.2 kb rat GnRH-R clone encodes a protein of 327 amino acids. A 1.3 kb clone encoding the mouse GnRH-R has previously been described (Tsutsumi et al., 1992). Although both the mouse and rat protein share significant homology with molecules belonging to the family of G protein-coupled receptors, they have certain unusual features, an example being the complete absence of a COOH terminal tail. The 3'-untranslated region reported missing in the mouse is present in the rat cDNA, where an extended 1 kb of 3'-untranslated region extending to the poly-A tail is shown. At the amino acid level, the rat GnRH-R shows considerable homology with that of the mouse. Electrophysiological studies with Xenopus oocytes and transfection of the cDNA into COS-1 cells, have shown that the 2.2 kb cDNA clone encodes a functional receptor.


Identification of regions in the rat serine dehydratase gene responsible for regulation by cyclic AMP alone and in the presence of glucocorticoids. Transcription of the rat serine dehydratase (SDH) gene is induced by glucagon, mediated by the action of cAMP. To identify the nucleotide sequences in the SDH gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5' flanking region of the rat SDH gene fused to the structural sequence encoding the bacterial reporter enzyme, chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat hepatoma cell line 7AD-7 were assayed by measuring CAT activity in the cell lysates. Chlorophenylthio-cyclic AMP (CPT-cAMP), a potent protein kinase A activating agent, stimulated the expression of SDH-CAT fusion genes, and these inductions could be enhanced further by the addition of dexamethasone, although the glucocorticoid alone had no effect on CAT activity. Deletion analysis demonstrated that an 80 bp region located approximately 3.5 kb upstream from the transcription initiation site of the rat SDH gene was responsible for stimulation of transcription by CPT-cAMP, whereas the 120 bp region immediately upstream of the cAMP responsive element (CRE)-containing sequences is essential for the enhancement of CPT-cAMP induction by the glucocorticoid.


Gonadotropin regulation of c-fos and c-jun messenger ribonucleic acids in cultured rat granulosa cells. c-Fos and c-jun are immediate early proto-oncogenes encoding proteins for the heterodimer AP-1, a DNA binding complex which regulates gene transcription. In order to investigate the presence and potential gonadotropin regulation of mRNAs for these proto-oncogenes in rat granulosa cells, we used Northern blotting of total RNA from cultured cells. Granulosa cells obtained from diethylstilbestrol (DES)-treated weanling rats were challenged with follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), dibutyryl cAMP ((Bu)2cAMP) or tetradecanoyl-13-phorbol acetate (TPA) either 2.5 h after cell isolation (day 0) or following a 2-day pretreatment with FSH (day 2). Freshly isolated cells treated with FSH exhibited 4-fold and 3-fold increases in c-fos and c-jun mRNAs, respectively, within 30 min. Two hours after FSH treatment, both c-fos and c-jun message levels diminished to near control levels. Granulosa cells pretreated for 2 days with FSH, then re-challenged with FSH, showed similar increases in both c-fos and c-jun messages. These effects were dose- and time-dependent on both day 0 and day 2. Likewise, (Bu)2cAMP also increased c-fos and c-jun mRNAs in a time- and dose-dependent manner on both day 0 and day 2. In contrast, LH or hCG minimally increased c-fos and c-jun mRNAs on day 0, but on day 2, both hormones markedly increased message levels in a manner similar to that seen with FSH. Analogous effects were observed with TPA which minimally stimulated c-fos and c-jun mRNAs on day 0, but markedly increased these messages on day 2. These studies demonstrate that c-fos and c-jun mRNAs can be induced in cultured rat granulosa cells by acute gonadotropin, (Bu)2cAMP or phorbol ester treatment and suggest that these immediate early proto-oncogenes may play a role in granulosa cell function.


Role of angiotensin II receptor subtypes on the regulation of aldosterone secretion in the adrenal glomerulosa zone in the rat. The role of AII receptors subtypes, AT1 and AT2, in the regulation of aldosterone secretion was studied in adrenal glomerulosa cells and membranes from rats on normal and low sodium intake, using AII receptor subtype-specific antagonists. In adrenal glomerulosa cells, more than 90% of the receptors were AT1 and there was a good correlation between the potencies of the antagonists to inhibit ligand binding, and AII-stimulated aldosterone production and inositol phosphate formation. The inhibition of basal and ACTH-stimulated cAMP by AII was also abolished by the AT1, but not the AT2, antagonist. Sodium restriction for 6 days increased both receptor subtypes in the same proportion, but only the AT1 antagonist inhibited AII-stimulated aldosterone production. The data demonstrate that AT1 receptor mediates the regulatory actions of AII in the adrenal zona glomerulosa.


Levocarnitine acetyl stimulates peripheral nerve regeneration and neuromuscular junction remodelling following sciatic nerve injury. The effects of levocarnitine acetyl were investigated on both peripheral nerve regeneration and neuromuscular remodelling in male Sprague-Dawley rats, three months of age, following crush of their left sciatic nerve. Levocarnitine acetyl, 150 mg/kg/day in drinking water, was given from one week before to 5, 15, 20, and 60 days after nerve crush. The sciatic nerve was examined morphologically at all given times and morphometrically at 15, 20, and 60 days after the lesion. Morphology, at 5, 15, and 60 days, and morphometry, at 60 days after the nerve crush, were also performed on the neuromuscular junction in the soleus and extensor digitorum longus muscles. Five days after nerve crush, complete axonal degeneration was observed in both control and treated rats. At 15 and 20 days, recovery from injury in treated animals was better than in controls, as shown by a significantly higher increase in the number of regenerating axons. At the same times, denervated endplates were present in both groups. At 60 days, axonal regeneration restored the number of axons to normal values in all injured animals, while their size maturation was greater in treated rats than in controls. A markedly lower number of degenerating elements was found in treated animals. In the neuromuscular junctions of the soleus and extensor digitorum longus muscles, nerve terminal branch points were reduced in the lesioned rats in comparison with uninjured ones. However, morphometric analysis revealed a greater endplate complexity in treated animals in which, at 60 days after nerve crush, nerve terminal branching and sprouting index values were significantly higher than in controls. It is concluded that levocarnitine acetyl exerts a beneficial effect on nerve regeneration processes and synaptic remodelling in crush-induced neuropathies.


Calcineurin trapped in liposomes inhibits the action potentials of isolated 3-days-old embryonic chick ventricle. Calcineurin, an intracellular protein phosphatase (type 2B), is reported to inhibit L-type (slow) calcium channels and thereby play a key role in channel inactivation. The present study was undertaken to examine effects of calcineurin on slow channel dependent action potentials of 3-days-old embryonic chick ventricle and to assess the role of this enzyme in regulation of developing slow channels. Calcineurin trapped in phosphatidylcholine-liposomes to facilitate its intracellular uptake was found to inhibit maximal upstroke velocity (+Vmax), overshoot and duration of action potentials. At higher doses of calcineurin containing liposomes the preparations ceased to exhibit spontaneous activity but elicited electrically driven action potentials with lower +Vmax and overshoot. These observations show that calcineurin down-modulates the embryonic cardiac slow channels under basal conditions.


The effect of chronic and acute haemorrhage on erythropoietin in the neonatal lamb. In all mammalian species studied the haematocrit (hct) declines after birth in the absence of any known nutritional deficiencies. The glycoprotein hormone, erythropoietin (Epo), is essential for normal red blood cell production. The aims of this study were 1) to investigate the changes in plasma Epo during the normal post-natal decrease in hct in lambs; 2) to compare the effects of chronic and acute haemorrhage in neonatal lambs; and 3) to test the hypothesis that the Epo response to haemorrhage is blunted in the neonatal period. Twenty-one lambs (0-9 weeks of age) were studied; group I (n = 8) were used to document normal post-natal changes (98 samples); group II (n = 7) lambs were haemorrhaged repetitively during weeks 3-6 (95 samples); group III (n = 6) lambs were bled once in the first 3-week period. In the group I (control lambs) the hct decreased from 30.6 +/- 1.3 (weeks 1 & 2) to a nadir of 23.2 +/- 0.8 (75.8% of initial value) in the 6th week, and the plasma Epo declined from 25.7 +/- 4.9 (week 1) to 12.3 +/- 1.0 mU/ml (week 6). In group II, the lambs were bled repetitively, a total of 510 +/- 32 ml blood being removed during weeks 3-6, the hct was 18.7 +/- 0.8 (81% of hct at nadir in controls) in week 6, and Epo was 26.9 +/- 13.3 in week 3, 23.4 +/- 3.6 mU/ml in week 6.(ABSTRACT TRUNCATED AT 250 WORDS)


[Cylindromas of the head and neck].

[Cylindromas of the head and neck]. The authors present the results of medical treatment in patients with head and neck cylindromas hospitalized in the Department of Otolaryngology. The Zeromski Hospital in Kraków, within the period 1961 to 1988.


[Malignant tumors of the head and neck in children. Part II]. 4 cases of malignant tumors of the head and neck diagnosed in our department in 1983-1987 are presented. They are: rhabdomyosarcoma (RMS) of the nose and the ethmoid sinuses, RMS of the nasal vestibulum, nasopharyngeal lymphoma malignum. Diagnostic difficulties were met by the histopathological evaluation of the tumors specimens, but the applied treatment was effective. There are no signs of recurrence during the observation period of 4-8 years.


[An outbreak of ovine granular keratoconjunctivitis of chlamydial origin, in Ivory Coast]. During the dry season 1990-1991, an outbreak of ovine granular keratoconjunctivitis occurred in Côte-d'Ivoire. The chlamydial etiology was demonstrated. All flocks were affected, with a morbidity rate of 30 to 70%. Lesions of keratitis were observed in 5 to 15% of the sick animals. The treatment with Auréomycine (ND-Specia, ophthalmic ointment) was constantly efficient.


[Quality control of the vaccines and cold chain during the national vaccination campaigns]. During the two vaccination campaigns against rinderpest and contagious bovine pleuropneumonia (CBPP) in 1989 and 1990, 507 vaccine samples were collected for quality control at each step of the distribution chain from the Veterinary Pharmacy of Abidjan to the vaccinator on the field. Parallel to that, the quality of the cold chain was checked. A total of 463 titrations were performed. All the titration levels were above the OIE/FAO standards, i.e. 10(2.5) DICT50/ml for rinderpest Valence and 10(7) viable germs/ml for contagious bovine pleuropneumonia Valence.


[Opioid receptors, tolerance and dependence]. Opioid substances act, both in the central and peripheral nervous systems, by regulating--via a specific receptor of the mu, delta or kappa type and a G protein--the activity of either of three recognized cellular effectors: adenylate cyclase, a K+ channel and a Ca++ channel. In the short term, opioid effects are of a neuromodulatory origin: they are likely to reflect opioid inhibition of neurotransmitter release (e.g.: of substance P in the spinal cord). In the long term, opioids induce the two adaptative phenomena known as tolerance and dependence whose mechanisms are considerably less well understood. Tolerance refers to a decreased responsiveness to the opioid upon repeated administration of the drug. Tolerance might reflect desensitization, a process which involves uncoupling of opioid receptor and of G protein (in the case of homologous desensitization) and, possibly, down-regulation of a G protein which the opioid receptor shares with other types of receptor (heterologous desensitization). Dependence refers to a latent, opioid-induced physiological state which expresses itself as a typical excitation syndrome upon withdrawal of the drug or administration of an opioid antagonist. The molecular events whereby dependence develops are unknown, yet they may involve (i) adenylate cyclase supersensitivity, an adaptation reaction where upon withdrawal of the inhibitory opioid induces an enhanced activity of the enzyme and, (ii) increased release of neurotransmitter (presynaptic facilitation).


[Analysis of the nucleotide sequence of a fragment (92-100%) of the CELO avian adenovirus genome]. The nucleotide sequence of 92-100% of the adenovirus CELO (FAV1), strain Phelps, genome has been determined. The computer analysis of the sequences revealed a ClaI site methylated by m*Ecodam. A recognition site for XbaI and two sites for PstI, not found in the corresponding genome of CELO, strain Ote, have been determined. Three extensive (more than 100 amino acid residues) open reading frames exist, coding for the polypeptides with molecular weights of 31.5, 19.3 and 14.5 kD (276, 178 and 128 amino acids, respectively). Some shorter open reading frames have been detected as well within the sequences studied.


[Imidazoline-guanidine site: a subtype of imidazoline receptors]. Since the demonstration that imidazoline and guanidinium alpha-2 adrenergic agonists induce some of their functional effects by a "nonadrenergic" mechanism, many efforts have been done to identify an imidazoline receptor. Binding studies have allowed to characterize two classes of potential imidazoline receptors: the "(p-amino)clonidine" and the "idazoxan" binding sites. These last, that we named "imidazoline-guanidinium receptive sites" (IGRS) on the basis of their ligand-recognition properties, have been identified, for the first time, in the proximal tubule from rabbit and human kidney. In the present report we will summarize the studies that led us to the characterization of IGRS.


[Chronopharmacology of fractionated heparin (nadroparin) administrated by subcutaneous route at prophylactic doses in healthy volunteers]. This study evaluated the effect of injection time on pharmacodynamic of a single subcutaneous bolus of nadroparine (7500 anti-Xa IC U) evaluated by anti-Xa activity (Hepaclot and Heptest) and by activated partial thromboplastin time (APTT by auto PTT reagent). 10 healthy male volunteers were studied at 4 different 24 hours periods with 4 different injection times (6 am, 12 am, 6 pm, 12 pm) and with a one week wash-out period between each period. No chronopharmacological variation of the anti-Xa activity evaluated by Hepaclot was found. However the anti-Xa activity evaluated by Heptest was higher at the sixth hour after 12 am injection (p = 0.0022). No difference on APTT values was observed whatever the injection time. So the injection time of nadroparine has a weak influence on anti-Xa activity and no effect on APTT; Before to conclude on the lack of chronopharmacological effect of nadroparine, it seems necessary to evaluate such a possibility with higher dosage, with sick and older subjects.


[Live enteroviral vaccines for the emergency nonspecific prevention of mass respiratory diseases during fall-winter epidemics of influenza and acute respiratory diseases]. The results of the 3-year controlled trials of a new method of nonspecific urgent prophylaxis of influenza and acute respiratory diseases (ADR) by immunization of healthy adults with standard live enterovirus oral vaccines, introduced in 2-3 administrations at intervals of 7-10 days, at the initial stages of autumn and winter epidemics are presented. Observations, carried out in three republics, covered more than 150,000 persons immunized with enterovirus interferonogenic vaccines. A considerable decrease in morbidity rate among the vaccinees was achieved (on the average, by 3.2 times) in comparison to that among nonimmunized subjects. The method of nonspecific prophylaxis with live enterovirus interferonogenic vaccines is recommended during outbreaks of diseases induced simultaneously by several causative agents of influenza and ARD, as well as by pathogenic enterovirus strains.


[The mechanism of the antiviral activity of an antiviral factor of a non-interferon nature]. Antiviral factor (AF) of protein nature has been isolated from chick embryo fibroblasts infected with Venezuelan equine encephalitis virus. The suppression of virus reproduction has been observed both in homologous and heterologous cell cultures when the preparation was introduced immediately after the adsorption of the virus after pretreatment of the cell monolayer. The study has demonstrated that the antiviral effect of AF is not linked with its IFN-alpha and TNF-alpha activity. Analysis of the results obtained in this study and earlier data contained in literature suggests that infected chick embryo fibroblasts release original cytokine of non-interferon nature with antiviral activity.


[Phosphatase and penicillinase activities as stable traits for the differentiation of the racial classification of Francisella tularensis]. In the causative agent of tularemia new markers correlating with different subspecies of this microbe have been detected. Thus, F. tularensis strains belonging to the American and Central Asian subspecies are characterized by phosphatase activity, which makes it possible to use the phosphatase test for their differentiation from the strains of the holarctic variety. F. tularensis subsp. mediasiatica are incapable of producing beta-lactamase which differentiates them from the representatives of the varieties holarctica and tularensis. These newly discovered signs are stable and do not depend on the virulence of the cultures under study and on the conditions of the cultivation of F. tularensis.


[Synovial sarcoma of the head and neck: a case report of parapharyngeal region and review of the literature]. Synovial sarcoma is a clinically and morphologically well defined entity that has been described extensively in Literature. It occurs primarily in the para-articular regions, usually in close association with tendon sheaths, bursae and joint capsules. On rare occasions it is also encountered in areas without any apparent relationship to synovial structures, as in the parapharyngeal region or the abdominal wall. It is considered the fourth most common type of sarcoma (7-10%) after malignant fibrous histiocytoma, liposarcoma and rhabdomyosarcoma. There are three histological variants: the classical biphasic, the monophasic fibrous type and the monophasic epithelial type (the biphasic and monophasic fibrous type are equally common). Clinical sign complaints are subtle and at times noted 20 years before diagnosis. The course of the disease is slow and insidious. The most typical presentation is that of a palpable deep-seated swelling or mass associated with pain or tenderness. Patients with synovial sarcoma in the head and neck (10%) tend to have difficulties in swallowing and breathing and not infrequently have alteration or loss of voice. Head and neck synovial sarcoma seem to originate from the paravertebral connective tissue spaces and manifest themselves as solitary retropharyngeal or parapharyngeal masses near the forking of the carotid. Additional cases in this general area have been reported in the soft palate, tongue, maxillofacial region, mandible corner, sternoclavicular region, scapular region and the cervical oesophagus. As in other types of sarcoma, the principal sites of metastases are the lung, but many make their appearance many years after the initial diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)


The structure basis of the poor fibrin specificity of urokinase (II)--The inhibition of urokinase A chain 149-157 on the fibrin stimulated activation of plasminogen by tissue type plasminogen activator. In view of the similarity of the charge distribution between fibrin A alpha 148-161 and A chain 149-157 of urokinase, the latter might compete with fibrin A alpha 148-161 when single chain pro-urokinase is converted to double chain urokinase. To test this, the stretch of urokinase A chain 135-157 was separated from the low molecular weight urokinase, a competitive binding between this stretch and fibrin to tPA kringle-2 was shown by radio-binding assay. The inhibition of the stretch on the fibrin stimulated activation of plasminogen was demonstrated in the caseinolytic system. The synthesized novapeptide urokinase A chain 149-157 (R-peptide) showed a significant inhibition on the activation of plasminogen in the presence of fibrin. By contrasting finely with R-peptide, a synthesized novapeptide in which Arg154 and Arg156 were replaced by Asp (D-peptide) did not show any inhibition effect on the fibrin stimulated activation of plasminogen by tPA. These results suggest that the positively charged residues in the stretch 149-157 of urokinase are crucial for the inhibition of fibrin binding with the kringle domain of urokinase.


Tempol and deferoxamine protect cultured rabbit lens epithelial cells from H2O2 insult: insight into the mechanism of H2O2-induced injury. In order to investigate the mechanism by which H2O2 damages the epithelium, 8 x 10(5) rabbit lens epithelial cells were treated with TEMPOL or deferoxamine and exposed to a single sublethal dose of 0.5 mM H2O2. TEMPOL is a SOD mimic, has a characteristic EPR spectrum and is metal independent. EPR spectra indicated that TEMPOL was not destroyed by H2O2, catalyzed the destruction of the superoxide anion, and penetrated the cells. Cells treated with H2O2 showed membrane blebbing, growth inhibition, an increase in GSSG, a dose-dependent decrease in GSH, ATP, NAD+, and in the activity of G3PDH, and in lactate production. H2O2 stimulated the hexose mono-phosphate shunt and induced single strand breaks in DNA. Treatment with TEMPOL or deferoxamine prevented or curtailed H2O2-induced inhibition of growth, the decrease in NAD+, the induction of single strand breaks in DNA, and membrane blebbing, but not the other biochemical parameters investigated. Both TEMPOL and deferoxamine prevent Fe+2-mediated generation of the damaging hydroxyl radical. TEMPOL reacts with superoxide and thus prevents it from recycling Fe+3 to Fe+2. It also oxidizes DNA-Fe+2 to DNA-Fe+3. Deferoxamine chelates intracellular Fe+3 and prevents its reduction to Fe+2. These compounds which limit the availability of Fe+2 by different means indicate that transition metals (including those bound to DNA) mediate certain of the damaging effects of H2O2.


Detection of free radicals in UV-irradiated lens by spin trapping ESR. We have made an ESR study on the UV-photolysis of lens and identified the origins of free radicals involved in the initial photochemical process by spin trapping technique. Two spin adducts were detected on irradiation of canine lens in the presence of a spin trapping reagent (DMPO); a spin adduct of sulfur centered radical derived from glutathione and the protonated adduct of hydrated electron. A free radical mechanism of initial photochemical injury in UV-irradiated lens was discussed, comparing with a photolysis of tryptophan plus cysteine solution.


Chorioretinitis caused by synthetic absorbable sutures. Two cases of chorioretinitis were caused by polymer material from the suture (Dexon) used in squint surgery. The lesions were macula edema, granulomatous uveitis and capillary occlusions on the ocular fundus located in the operated area. To confirm the cause of this injury, an immunological investigation was performed. Macrophage block and an incomplete adjuvant were necessary to lead delayed hypersensitivity against the synthetic polymer polyglycolic acid (PGA). The mice showed a strong reaction as hyperemia and swelling on their experimental footpads after the injection of PGA. Histologically the footpad accumulated a large quantity of lymphocytes in the swollen tissue but the control footpad had no reaction.


[Effect of Zea pollinium on the structure and function of erythrocyte membrane in rats]. The aged-related changes of ATPase activity as well as the contents of MDA, sulfhydryl and sialic acid of erythrocyte membrane had been observed in many studies. In this paper, the effect of Zea pollinium on the structure and function of erythrocyte membrane in rats was observed. 12 male rats were divided randomly into pollen and control groups, the former was fed with diet containing 10% Zea pollinium; while no pollen for the latter. After feeding for 10 weeks, the Na+, K(+)-ATPase and Ca2+, Mg(2+)-ATPase activities were 311.5 +/- 35.5 and 813.8 +/- 43.4 nmolPi/mg protein.h respectively in pollen group, significantly higher than that of the control group (209.9 +/- 23.9 and 624.9 +/- 23.3 nmolPi/mg protein.h). The contents of sulfhydryl and sialic acid were also increased, but the content of MDA was markedly decreased with the use of Zea pollinium. These results indicated that Zea pollinium could inhibit formation of lipid peroxidates, protect the structure and function of erythrocyte membrane from the injury of peroxidate.


Hospital-based nursing response to perinatal drug exposure. This study examined hospital-based nursing interventions used for the problem of drug exposure in newborns and their mothers. Although there are few published reports of treatment services for this at-risk population, there is a consensus among health care providers on the need for treatment and the type of services that a treatment program should offer. A questionnaire measured use of assessment tools for newborn drug exposure and maternal drug use, patterns of referral, and use of aftercare and community treatment programs. The mean number of policies was 2.5, and the mean number of services offered was 1.042, consisting mostly of referral to community programs after discharge. Results can be used to develop quality assurance programs to address this issue and provide a more comprehensive response to the problem.


Postoperative follow-up: tracking compliance and complications. The focus of ambulatory surgery is wellness. For the ambulatory surgery client, most of postoperative recovery occurs at home, away from close nursing supervision. Postoperative follow-up is essential after discharge to track the client's compliance with discharge instructions and to identify complications. This presents a unique challenge and opportunity for nurses to evaluate the effectiveness of their teaching, identify quality improvement issues, and detect complications early during the recovery period.


Inactivation of both APC alleles in an early stage of colon adenomas in a patient with familial adenomatous polyposis (FAP). To examine whether the dosage effect of germ-line mutations in patients with familial adenomatous polyposis (FAP) is sufficient to cause colorectal adenomas, or an additional somatic mutation of the normal allele is required as well, we have investigated somatic mutations of the APC gene in multiple adenomas developed in one FAP patient. In addition to a 5-bp deletion of one allele present constitutionally in this patient, the normal APC allele had been lost in five of seven DNA samples extracted from small adenomas (< 3 mm in diameter) with mild or moderate atypia. This result indicates that the inactivation of both alleles of the APC gene is probably essential for the development of an early-stage adenoma, in agreement with the two-hit mutational model underlying the concept of tumor suppressor genes.


Screening for germ-line mutations in familial adenomatous polyposis patients: 61 new patients and a summary of 150 unrelated patients. We report here the result of a screening for germ-line mutations in the adenomatous polyposis coli (APC) gene in 61 new familial adenomatous polyposis (FAP) patients as well as a summary of the results of 150 patients. Examination of the entire coding region of the APC gene, based on a ribonuclease protection assay coupled with the polymerase chain reaction (PCR), disclosed mutations that were considered to cause significant defects in the APC product in 97 of 150 unrelated FAP patients. Our findings revealed the following characteristics of the germ-line mutations of APC: 1) the great majority of the mutations were found to truncate the APC product; 2) almost all of the mutations were located within the first half of the coding region; 3) no correlation was observed between the locations of germ-line mutations and extracolonic manifestations in FAP patients; 4) more than 80% of base substitutions in the APC gene were from cytosine to other nucleotides, nearly one-third of which occurred at the GpG site. Our results provide information helpful to an understanding of the APC gene and will also contribute to presymptomatic diagnosis of members in FAP families.


Linkage studies and mutation analysis of the PDEB gene in 23 families with Leber congenital amaurosis. The phenotype in the rd mouse is similar to the clinical presentation of Leber congenital amaurosis (LCA) in humans. Recently a nonsense mutation in the beta subunit of the cGMP phosphodiesterase (Pdeb) gene has been defined as the cause for the rd phenotype in the mouse and has raised the question as to whether mutations in the human PDEB gene might cause LCA. We have previously cloned and characterized the human homologue of the mouse Pdeb gene and have mapped it to chromosome 4p16.3. In this study, a total of 23 LCA families of various ethnic backgrounds have been investigated. Linkage analysis using highly polymorphic (CA)n microsatellites has excluded the PDEB gene as a cause for LCA in 6 families. In the remaining 17 families, we have searched for mutations in the 22 exons of the PDEB gene using single-strand gel electrophoresis (SSGE). Multiple exonic polymorphisms have been determined. However, no DNA changes in the PDEB gene have been identified in our study population which could be causative for the LCA phenotype.


Absence of linkage between the angiotensin converting enzyme locus and human essential hypertension. The angiotensin converting enzyme (ACE) is a key component of the renin angiotensin system that contributes to the regulation of blood pressure (BP). Recent demonstration of linkage between the ACE locus and elevated BP in a rat model of hypertension has further emphasized ACE as a candidate gene in human hypertension. We report the localization of the ACE gene on the genetic map of chromosome 17, and identify an extremely polymorphic marker at the human growth hormone (hGH) locus which shows no recombination with ACE. We have found no evidence to support linkage between the ACE locus and hypertension, which suggests that mutations at the ACE locus do not commonly contribute to the pathogenesis of hypertension in our test population.


Exclusion of DNA changes in the beta-subunit of the c-GMP phosphodiesterase gene as the cause for Huntington's disease. To identify expressed sequences within candidate regions for the Huntington's disease (HD) gene in 4p16.3, we isolated the gene encoding the beta subunit of the human cGMP phosphodiesterase (PDEB). We formally assessed this as a candidate gene for HD based on it's expression in brain, the demonstration of linkage disequilibrium between intragenic DNA markers and HD, and the demonstration that mice with a mutation in this gene have a reduction of neurons in particular brain regions. We investigated all 22 exons of PDEB and 5'-flanking region for point mutations in 16 HD patients of different ethnic origins using single strand conformational polymorphism analysis. The underlying DNA changes found initially exclusively in HD patients were excluded as the cause for HD.


Maternal but not paternal transmission of 15q11-13-linked nondeletion Angelman syndrome leads to phenotypic expression. Angelman syndrome (AS) may result from either maternally inherited deletions of chromosome 15q11-13 or from paternal uniparental disomy for chromosome 15. This is in contrast to Prader-Willi syndrome (PWS), which is caused by either paternal deletion of this region or maternal disomy for chromosome 15. However, 40% of AS patients inherit an apparently intact copy of chromosome 15 from each parent. We now describe a family in which three sisters have given birth to four AS offspring who have no evidence of deletion or paternal disomy. We show that AS in this family is caused by a mutation in 15q11-13 that results in AS when transmitted from mother to child, but no phenotype when transmitted paternally. These results suggest that the loci responsible for AS and PWS, although closely linked, are distinct.


Expressed genes, Alu repeats and polymorphisms in cosmids sequenced from chromosome 4p16.3. The sequences of three cosmids (90 kilobases) from the Huntington's disease region in chromosome 4p16.3 have been determined. A 30,837 base overlap of DNA sequenced from two individuals was found to contain 72 DNA sequence polymorphisms, an average of 2.3 polymorphisms per kilobase (kb). The assembled 58 kb contig contains 62 Alu repeats, and eleven predicted exons representing at least three expressed genes that encode previously unidentified proteins. Each of these genes is associated with a CpG island. The structure of one of the new genes, hda1-1, has been determined by characterizing cDNAs from a placental library. This gene is expressed in a variety of tissues and may encode a novel housekeeping gene.


Herpesvirus vector gene transfer and expression of beta-glucuronidase in the central nervous system of MPS VII mice. Genetic disorders affecting the central nervous system (CNS) can potentially be treated by gene transfer using vectors which infect and express genes in post-mitotic neurons. Herpesviruses establish latent infections in neurons during which only one viral gene (LAT) is expressed, thus the LAT promoter may express foreign genes in latently infected CNS cells. Expression of a beta-glucuronidase gene driven by the LAT promoter was tested in mice lacking this enzyme, which are a model for a human genetic disease affecting the CNS (mucopolysaccharidosis VII, Sly disease). Cells expressing the missing enzymatic activity were present in the trigeminal ganglia and brainstems of latently infected animals, up to four months post-inoculation, demonstrating the potential of this approach for the long-term expression of foreign genes in the CNS.


Pattern of somatic transposition in a high copy Ac tomato line. A transgenic tomato line containing between eight and ten copies per genome of an exceptionally active maize transposable element Ac has previously been described. Southern analyses indicated that these elements are somatically active in these plants. In order to characterize further the pattern of somatic transposition in this line, 24 independent Ac insertion events from a single plant were cloned. In 21 cases, Ac inserted into single copy genomic DNA while in three cases Ac inserted into sequences present at two to four copies per genome; none of the insertions occurred into more highly repetitive DNA. The chromosomal locations of 20 insertion sites were determined by RFLP mapping and a pattern of small dispersed clusters emerged. Thirteen of the 20 insertion sites were linked to at least one other insertion site but these were distributed over nine of the 12 tomato chromosomes. Only one Ac insertion was linked to the T-DNA locus. The structural integrity of these Ac elements was examined and no evidence of deletions or other rearrangements suggestive of Ds elements was found. The implications of these findings with respect to the use of Ac as a transposon tag in heterologous species are discussed.


Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue-specific expression of a legumin gene. A 2.4 kb fragment containing the 5'-flanking region and the 5'-noncoding sequence of the Vicia faba legumin gene LeB4 mediates high level seed-specific expression in transgenic tobacco plants. Deleted derivatives of this legumin upstream sequence were fused to the npt-II reporter gene to determine the tissue-specific activity of the chimeric constructs in stably transformed tobacco plants. The results indicate the presence of positive regulatory, enhancer-like cis elements within 566 bp of the upstream sequence. Most importantly, however, these elements are only fully functional in conjunction with the core motif CATGCATG of the legumin box around position -95, since destruction of the motif by a 6 bp deletion in an otherwise intact 2.4 kb upstream sequence drastically reduces expression in seeds. At the same time, low level expression in leaves is observed. The occurrence of similar CATGCATG consensus cis elements with alternating purine and pyrimidine base pairs in front of several other plant genes suggests a functional role of the motif in a wider range of plant promoters.


[Silica urinary calculi. Report of a case]. The authors report a case of anuria due to bilateral ureteric obstruction by silica stones in a patient taking magnesium trisilicate for many years. A review of the literature concerning silica stones is presented. The diagnosis should be suggested by the clinical history and confirmed by stone analysis. The treatment of silica stones is not specific, but must be associated with withdrawal of the drug responsible.


[Treatment of retinoblastoma using accelerated protons]. Thanks to the Bragg vertical and the straight course of protons, the physical selectivity of proton beam is greater then that of megavoltage photons. Since January 1991 we treated 3 retinoblastomas with proton beam at the cyclotron of Louvain-la-Neuve. Results are encouraging. The short term outcome seems comparable to phototherapy, but the long term expectancy is better, because of reduced secondary malignancies. Proton beam treatment for selected retinoblastomas seems a good alternative to external radiotherapy.


ACE inhibition in the tissues.

ACE inhibition in the tissues. The discovery of the components of the renin angiotensin system (RAS) in various tissues gave rise to the idea that functional "tissue" renin-angiotensin systems exist that are more or less independent of the hormonal RAS. Further support to this notion came from the recent demonstration of the mRNA for the protein components of the RAS in a number of organs. Last not least, the introduction of the CE inhibitors, generating an enormous scientific interest in the role of the RAS, has contributed to the concept of "tissue" RAS, since some of the effects of these drugs were thought to be reconciled better with "tissue" than with plasma RAS inhibition. However, this model of plasma vs "tissue" RAS still suffers from a number of conceptual problems that have to be dealt with. For instance, with respect to the "tissue" RAS in the vascular wall it is not clear at present whether ACE is at all active inside endothelial cells. In addition, all evidence available speaks against its localisation in the vascular media, while there may be some activity of the enzyme in the adventitial layer. Concerning the heart, there is at present no unequivocal evidence for a local ANG II production through angiotensin converting enzyme (ACE) in cardiac tissue outside the coronary vessels. The localisation of ANG II generation within tissue RAS in the adrenal gland or in the kidney, where ANG II may be generated through CE at the tubular brush border, is far from being elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)


[Antidotal effects of 2,3-dimercaptopropane-1-sulfonate sodium (DMPS) and combined with diazepam on acute poisoning caused by sodium ammonium dimethyl-2-propano-1,3-dithiosulfate monohydrate (SCD)]. In mice, DMPS (250 mg/kg, i.v.) combined with diazepam (1.25 mg/kg, i.p.) could increase LD50 of p. o. SCD 5.3 times. DMPS (62.5 mg/kg, i.v.) antagonized completely the respiratory depression and neuromuscular blockade caused by SCD(7.5 mg/kg, i.v.) in rabbits. SCD (15 mg/kg, i.v.) caused tremor, tonic convulsion and the abnormal paroxysmal discharges in EEG in rabbits. DMPS (0.5 mg/kg, i.c.v) could not eliminate the abnormal paroxysmal discharges in EEG of rabbits. DMPS (62.5 mg/kg, i.v.) combined with diazepam (5 mg/kg, i.v.) completely and rapidly antagonize these toxic symptoms and the abnormal changes in EEG.


Identification of two promoters within human cytomegalovirus morphologic transforming region II. A 980-bp subfragment of human cytomegalovirus (HCMV) strain Towne has been previously identified as morphologic transforming region II (mtrII) because of its ability to induce focal transformation of NIH 3T3 cells. Transcripts from this region, which could encode the three open reading frames (ORFs), 79, 83, and 34 amino acids (aa), detected by DNA sequence analysis, are expressed early during HCMV infection. In this report, the mRNA start sites for promoters (P1 and P2) were mapped within Towne mtrII by primer extension using RNAs isolated from transformed NIH 3T3 cells. The Towne mtrII promoters exhibited similar activities to the SV40 enhancerless early promoter. Equivalent promoter activities were detected within the mtrII colinear nontransforming region from HCMV strain Tanaka. Two subclones of Towne mtrII (5' 440-bp and 3' 540-bp), each containing one promoter, were generated utilizing a unique BgII site which also interrupted the 79-aa ORF. In transfection assays, neither the 5' 440-bp promoter subclone containing a truncated 79-aa ORF nor the 3' 540-bp subclone containing intact 83- and 34-aa ORFs exhibited transforming activity. These data indicated that transformation by HCMV mtrII did not occur by promoter insertion. The identification of these early promoters will allow further studies on the regulation of important HCMV early genes known to be involved in viral/host interactions.


Candid No. 1 Argentine hemorrhagic fever vaccine protects against lethal Junin virus challenge in rhesus macaques. The protective efficacy of Candid No. 1, a live-attenuated vaccine against Argentine hemorrhagic fever (AHF), was evaluated in non-human primates. Twenty rhesus macaques immunized 3 months previously with graded doses of Candid No. 1 (16-127, 000 PFU), as well as 4 placebo-inoculated controls, were challenged with 4.41 log10 PFU of virulent P3790 strain Junin virus. All controls developed severe clinical disease; 3 of 4 died. In contrast, all vaccinated animals were fully protected; none developed any signs of AHF during a 105-day follow-up period. Viremia and virus shedding were readily detected in all placebo-vaccinated controls, while virus could be recovered only once (by amplification) from throat swabs of 2 Candid No. 1 vaccinees on day 21. Vigorous secondary-type neutralizing and immunofluorescent antibody responses were seen in most vaccinees that had received 3 log10 PFU Candid No. 1 or fewer; all others, including those receiving 127,200 PFU, maintained relatively stable titers during follow-up. Candid No. 1 was highly immunogenic and fully protective against lethal Junin virus challenge in rhesus macaques, even at extremely low (16 PFU) vaccine doses.


Prostaglandin E2 stimulates adrenocorticotrophin and cortisol secretion via a hypothalamic site of action in fetal sheep. The hypothesis that prostaglandins stimulate fetal adrenocortical activity via a central site of action within the fetal brain was tested in chronically catheterized fetal sheep. At day 120 gestation (term = 145 days) fetal sheep were surgically prepared with catheters in the lateral cerebral ventricle, jugular vein and carotid artery and experiments began five days later. Intravenous (i.v.) infusion of prostaglandin E2 (30 or 120 micrograms.h-1) caused a significant dose-related increase in fetal plasma concentrations of ACTH. Despite this increase in ACTH, cortisol was only stimulated after the highest dose of prostaglandin E2. Intracerebroventricular (i.c.v.) infusion of PGE2 (30 micrograms.h-1) also stimulated ACTH secretion although the peak response was delayed and considerably less compared with the same dose administered intravenously. Prostaglandin F2 alpha administered i.v. or i.c.v. had no effect on circulating concentrations of either ACTH or cortisol. These data provide evidence that prostaglandin E2 can stimulate fetal ACTH secretion by acting in the fetal brain. Furthermore, the greater release of ACTH after i.v. compared with i.c.v. prostaglandin E2 suggests that a site of action other than the brain, such as the pituitary gland, may also be important. These results provide further evidence that during late gestation circulating prostaglandins can act to stimulate fetal pituitary-adrenal maturation.


[Wilms' tumor. Its multidisciplinary management]. A total of 115 children with a histopathological diagnosis of Wilms' tumor were studied. The average age was three years. An abdominal tumor was the most frequent clinical manifestations, with a predominating clinicopathological stage II. The most important prognostic factors were the clinical stage and histological subvariety. A five year disease free period during the early stages was very favorable. On the other hand, advances stages and unfavorable histopathology established a poor prognosis. In our experience, stages I and II and favorable histology should not receive radiotherapy but instead brief chemotherapy. The global five year survival was 82%. All the patients with an unfavorable histology occupied stages II and IV. a comparison of disease free survival between stages I and II against III and IV showed statistical significance (p 0.01). Statistical significance also appeared upon comparison between unfavorable versus favorable (p 0.01) histology. Emphasis is placed upon multidisciplinary management of this type of malignant neoplasias.


GTP gamma S effects on phosphatidylinositol phospholipase C alpha isoenzyme activity isolated from guinea pig uterine smooth muscle at different stages of pregnancy. The ability of GTP gamma S to activate release of inositol polyphosphates from isolated permeabilised guinea pig uterine smooth muscle cells and from partially purified PI-PLC alpha has been studied. Streptolysin O permeabilised and [3H]inositol prelabelled cells show a time dependent release of inositol polyphosphates, predominantly inositol 4-phosphate. Ca2+ stimulated IP release with a Ka of 161 +/- 1.1 nM and this was further enhanced in an additive manner by GTP gamma S between 1-100 microM; the Ka for Ca2+ in the presence of 0.1 mM GTP gamma S was 117 +/- 0.7 nM. GTP gamma S activation of IP production did not require Ca2+ in the medium. Permeabilisation of the uterine smooth muscle cells with Streptolysin O readily released PI-PLC activity into the medium. However, unlike studies with isolated membranes 63.4 +/- 6.4% of the enzyme activity remained associated with membranes and/or particulate fractions of the cell. Studies were undertaken with PI-PLC alpha, the predominant isoenzyme form, partially purified from uterine smooth muscle at different stages of pregnancy by Q-Sepharose and Heparin-Agarose chromatography. The enzyme co-purifies with firmly associated GTP-binding activity. Enzyme prepared from near-term uterus is activated by 0.1 mM GTP gamma S, up to 100% when Ca2+ is between 0.1-1 microM, while 10 microM AlF4- under those conditions caused complete inhibition of the enzymes. Responses for enzymes prepared from non-pregnant uteri were broadly similar. In contrast enzyme preparations from guinea pig uteri at 20-60 days of pregnancy show an inhibition of activity in response to 0.1 mM GTP gamma S addition.(ABSTRACT TRUNCATED AT 250 WORDS)


The ratio of plasma bioactive to immunoreactive ACTH-like activity increases with gestational age in the fetal lamb. The fetal ovine pituitary-adrenal axis plays an important role in the timing of parturition, in fetal lung maturation, and in fetal and neonatal responses to stress. While the ovine pituitary during the last third of gestation (term = 145 days) is capable of secreting immunoreactive ACTH (iACTH) in response to various stimuli, plasma cortisol levels frequently do not reflect the rise in plasma ACTH. Therefore, we examined the relationship between plasma iACTH and steroidogenic ACTH-like activity (bACTH) in a group of immature fetal lambs (Group I: gestational age = 97 +/- 2 days, mean +/- SEM, n = 16) and a group of near-term fetuses (Group II: gestational age = 136 +/- 1 days, n = 13) following acute exteriorization. Plasma iACTH was determined by RIA. Plasma bACTH was determined by the ability of glass-extracted material to stimulate corticosterone (B) production in an acutely dispersed rat adrenal bioassay. Plasma iACTH and bACTH levels varied among animals within age groups, with iACTH tending to be higher in immature fetal lambs (Group I) than near-term lambs (Group II) and bACTH being higher (P < 0.05) near term than earlier (Group I: iACTH = 807 +/- 273 pg/ml, bACTH = 173 +/- 44 pg/ml; Group II: iACTH = 405 +/- 85 pg/ml, bACTH = 371 +/- 96 pg/ml). The proportion of iACTH that had biologic activity (e.g. B/I ratio) was significantly greater in the older than in the younger fetuses (Group II: B/I = 0.862 +/- 0.109; Group I: B/I = 0.462 +/- 0.105 P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Dependence of release of [3H]noradrenaline from rabbit pulmonary artery on internal sodium. 1. [3H]Noradrenaline ([3H]NA) release from the isolated main pulmonary artery of the rabbit has been measured in the presence of uptake blockers (cocaine, 3 x 10(-5) M, and corticosterone, 5 x 10(-5) M) and after blocking the monoamine oxidase enzyme by pargyline (1.2 x 10(-4) M). 2. In normal Krebs solution Mn2+ (2 mM) significantly inhibited both [3H]NA release (approximately 80%; P < 0.001) and the contraction following 2 Hz field stimulation. 3. In Ca(2+)-free, EGTA (1 mM)-containing solution, the Na+ pump was inhibited by removal of K+ from the external medium. In Na+ pump-inhibited arteries, 2 mM Mn2+ (free Mn2+, 1 mM) increased the spontaneous release of [3H]NA according to the time of Na+ loading. TTX (10(-7) M) did not inhibit significantly the Mn(2+)-induced [3H]NA release from Na(+)-loaded preparations (percentage inhibition, approximately 24; P > 0.30). 4. Without Na+ loading (Ca2+ free, EGTA alone), Mn2+ failed to promote 3H release from arteries. 5. With constant Na+ loading (120 min 'K(+)-free' perfusion in Ca(2+)-free, 1 mM EGTA-containing solution), the release of 3H was also directly dependent on free Mn2+ concentration (0.2, 0.6 and 1 mM). 6. The Mn2+ (2 mM; free Mn2+, 1 mM)-induced 3H release from Na(+)-loaded nerves (120 min 'K(+)-free', perfusion) was further enhanced, when external Na+ was simultaneously reduced from 139.2 to 26.2 mM (choline+ or sucrose substitution). 7. Diphenylhydantoin (DPH, 10(-4) M) significantly reduced the Mn(2+)-evoked 3H release (approximately 44%; P < 0.02) when it was present during 'K(+)-free', perfusion. 8. Mn2+ was ineffective in releasing 3H if the Na+ pump was previously reactivated by readmission of K+ to Na(+)-loaded arteries. 9. It is concluded that in Ca(2+)-free solution Mn2+ releases neurotransmitter in a manner which depends on the degree of loading with internal Na+. The results suggest this depends at least partly on a block of Ca2+ efflux.


Action of brief pulses of glutamate on AMPA/kainate receptors in patches from different neurones of rat hippocampal slices. 1. Outside-out patches were isolated from granule cells of dentate gyrus and pyramidal cells of CA3 and CA1 regions of rat hippocampal slices. Patches were exposed briefly to L-glutamate using a piezo-driven double-barrelled application pipette. 2. Applications of glutamate (1 mM) of 1 ms duration activated patch currents which rose and decayed rapidly. The 20-80% rise time of these glutamate receptor (GluR)-mediated currents was usually 0.2-0.6 ms. At -50 mV the peak current varied from 10 to 500 pA in different patches. 3. The peak current-voltage relation for brief pulses of 1 mM glutamate was virtually linear in normal extracellular solution for patches from the three cell types (-100 to 60 mV). 4. The permeability of GluR channels activated at the peak to Ca2+, relative to K+, was less than 0.1 for all three cell types (under bi-ionic conditions with Ca2+ on the extracellular side and K+ on the intracellular side of the membrane). 5. The offset decay time constant of the current following 1 ms pulses of 1 mM glutamate was brief, with mean values of 3.0 +/- 0.8, 2.5 +/- 0.7, and 2.3 +/- 0.7 ms for dentate, CA3 and CA1 cell patches, respectively. Offset time constants were independent of membrane potential and independent of glutamate concentration (200 microM and 1 mM) for the three cell types. 6. Applications of 1 mM glutamate of 100 ms duration showed that glutamate responses desensitized rapidly. The time constants for desensitization were 9.4 +/- 2.7, 11.3 +/- 2.8, and 9.3 +/- 2.8 ms for patches from dentate, CA3 and CA1 cells respectively. Desensitization time constants were only weakly dependent on glutamate concentration (200 microM and 1 mM) for the three cell types. Thus offset time constants are about four times faster than desensitization time constants for both glutamate concentrations. 7. Double pulse application of glutamate indicated that even a 1 ms pulse of 1 mM glutamate causes partial (about 60%) desensitization of GluR channels. The time course of recovery from desensitization was slower in dentate gyrus granule cell patches than in CA3 or CA1 pyramidal cell patches. 8. Desensitization was studied at equilibrium by exposing patches to low glutamate concentrations for at least 15 s before a 1 ms test pulse of 1 mM glutamate.(ABSTRACT TRUNCATED AT 400 WORDS)


Effect of catecholamines on intracellular pH in sheep cardiac Purkinje fibres. 1. It has been reported that catecholamines affect intracellular pH (pHi) in a number of tissues, generally by altering the kinetics of the Na(+)-H+ exchanger. We postulated that catecholamines might affect pHi in cardiac tissue. We tested this in resting sheep cardiac Purkinje fibres by measuring transmembrane potential and pHi with standard and H(+)-sensitive microelectrodes. 2. Adrenaline and the beta-adrenergic agonist isoprenaline, both 5.0 x 10(-6) M, resulted in depolarization and intracellular acidification (adrenaline, 0.03 +/- 0.01 pH units, n = 8, P = 0.005; isoprenaline, 0.08 +/- 0.01 pH units, n = 17, P = 0.0001). The alpha-adrenergic agonist phenylephrine, at concentrations up to 200 microM, had no significant effect on membrane potential or pHi. 3. Isoprenaline significantly attenuated the half-time (t0.5) for pHi recovery from intracellular acidification induced via the NH4Cl pulse technique. Isoprenaline also attenuated the hyperpolarization that is normally seen at the onset of pHi recovery. Phenylephrine slightly reduced the t0.5 for recovery, although the reduction did not reach statistical significance. 4. Forskolin, 7.5-10 x 10(-5) M, an agent that raises intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP), also induced depolarization and acidification, similar to that induced by adrenaline and isoprenaline. 5. In the presence of the Na(+)-H+ exchange blocker 5-dimethyl amiloride, 2-6 x 10(-5) M, isoprenaline-induced acidification was blunted but not abolished. When administered in Na(+)-free Tyrode solution, isoprenaline-induced acidification was also not abolished. Buffering power, tested using the NH4Cl method, was not decreased by isoprenaline, but rather, was slightly increased. Reversal of H+ driving force across the cell membrane from the normally inward direction to outward (achieved by increasing pHo to 8.3-8.5 and depolarizing the membrane with 10 mM K+ solutions) did not prevent intracellular acidification from occurring in the presence of isoprenaline. When glycolysis was inhibited by a 60 min exposure to glucose-free solution containing 5.5 mM 2-deoxyglucose, acidification by isoprenaline was nearly abolished. 6. We conclude that, in resting sheep Purkinje fibres, beta- but not alpha-adrenergic stimulation results in intracellular acidification and depolarization, probably mediated via an increase in cyclic AMP. beta- but not alpha-adrenergic stimulation slows the rate of recovery from intracellular acidification and blunts the hyperpolarization associated with this recovery. 7. The intracellular acidification appears to be due both to partial inhibition of Na(+)-H+ exchange and to stimulation of glycolysis by beta-adrenergic agents.


Prostaglandin modulation of Ca2+ channels in rat sympathetic neurones is mediated by guanine nucleotide binding proteins. 1. The effects of prostaglandins on whole-cell Ca2+ currents of acutely isolated and short-term cultured adult rat superior cervical ganglion neurones were investigated using the patch-clamp technique. 2. Prostaglandin E2 (PGE2) produced a rapid, reversible and concentration-dependent reduction of the sympathetic neurone Ca2+ current. The effects of PGE2 were both voltage and time dependent. The relationship between Ca2+ current inhibition and test potential was 'bell' shaped with maximal inhibition occurring near the potential where the Ca2+ current amplitude was maximal (ca + 10 mV). In the presence of PGE2, the Ca2+ current rising phase was slower and biphasic at potentials between 0 and +40 mV. 3. Prolonged (> 2 min) application of 1 microM PGE2 resulted in a desensitization of the response. Similarly, repeated short (ca 1 min) applications of 1 microM PGE2 resulted in a progressive tachyphylaxis of the response. 4. A concentration-response curve for PGE2 was well described by a single-site binding isotherm. The concentration producing half-maximal block (IC50) and the maximal attainable reduction of the Ca2+ current were 7.8 nM and 48%, respectively. 5. When compared at a concentration of 1 microM, PGF2 alpha was less potent (33% inhibition) than PGE2 but otherwise produced similar effects. In contrast, 1 microM PGD2 had negligible effects. 6. Activation curves, as derived from tail current amplitudes, were described by the sum of two Boltzmann functions in both the presence and absence of PGE2. In the presence of PGE2, the activation curve was shifted toward more depolarized potentials. Most of the shift could be accounted for by a decrease in the fractional amplitude of the current component activated at hyperpolarized potentials along with a concomitant increase in the component activated at depolarized potentials. The deactivation time constant (0.33 ms), measured at -40 mV, was not altered by PGE2. 7. The majority of the Ca2+ current inhibition produced by PGE2 was relieved by depolarizing conditioning pre-pulses to +80 mV for 50 ms. 8. Dialysis of sympathetic neurones with a pipette solution containing 2.0 mM guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) abolished the effects of PGE2 on the Ca2+ current. Pretreatment of the neurones overnight with pertussis toxin significantly, but incompletely, decreased the Ca2+ current inhibition produced by PGE2. 9. The prolonged Ca2+ tail current component induced by the dihydropyridine Ca2+ channel 'agonist' (+)202-791 (2 microM) was unaffected by 1 microM PGE2. 10. PGE2 partially inhibited the Ca2+ current component remaining after pretreatment of the neurones with 10 microM omega-conotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)


Adrenaline and extracellular ATP switch between two modes of acid extrusion in the guinea-pig ventricular myocyte. 1. Intracellular pH (pHi) was recorded in isolated guinea-pig ventricular myocytes using the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphthorhodafluor). 2. Addition and removal of 10 mM NH4Cl was used to induce an intracellular acid load in a myocyte perfused with HCO3(-)-buffered solution containing amiloride. Under these conditions, subsequent pHi recovery is known to rely upon Na(+)-HCO3- co-transport into the cell. The application of 0.5-5 microM adrenaline resulted in an inhibition of this pHi recovery. 3. In HEPES-buffered solution, where acid extrusion is mediated primarily by Na(+)-H+ antiport, pHi recovery from an acid load was stimulated by the application of adrenaline. 4. In HCO3-/CO2-buffered solution (no amiloride), when both acid-aquivalent extruders are activated by an intracellular acidification, adrenaline was found to slow pHi recovery. 5. When both carriers were inhibited in Na(+)-free, HCO3(-)-buffered medium, adrenaline had no effect on pHi, ruling out any effect of the catecholamine on background acid loading. 6. The voltage clamp technique was used to test if the inhibitory effect of adrenaline on amiloride-resistant, HCO3(-)-dependent pHi recovery was due to an efflux of HCO3- ions through catecholamine-activated anion channels. During pHi recovery, membrane depolarization, sufficient to reverse the electrochemical driving force acting on HCO3-, had no effect upon pHi recovery rate. 7. The above results show that adrenaline has direct but opposite effects on Na(+)-HCO3- co-transport and Na(+)-H+ antiport. In the presence of this agonist, the pHi dependence of Na(+)-HCO3- symport was shifted to the left along the pHi axis by 0.13 +/- 0.03 units (n = 4) whereas that for Na(+)-H+ antiport was shifted in the opposite direction by only 0.07 +/- 0.01 units (n = 3). Following an acid load, the net effect of adrenaline under physiological conditions was, therefore, a slowing of pHi recovery. 8. The application of extracellular ATP (ATPo, 10-50 microM) mimicked the effects of adrenaline on both Na(+)-H+ exchange and Na(+)-HCO3- symport. 9. Adenosine (50 microM) and ADP (50 microM) did not induce any inhibition of Na(+)-HCO3- symport, suggesting that the inhibition induced by ATP was not mediated through P1 or P2-purinergic receptors. 10. We conclude that Na(+)-H+ antiport and Na(+)-HCO3- symport are both coupled to adrenaline and ATPo receptors. Activation of these receptors switches acid-equivalent extrusion from a situation dependent on both HCO3- and H+ ions to one nearly exclusively dependent upon H+.


Shear stress-induced calcium transients in endothelial cells from human umbilical cord veins. 1. Changes of the free cytosolic Ca2+ concentration induced by shear stress were measured in Fura-2 acetoxymethyl ester-loaded endothelial cells from human umbilical cord veins. 2. We were able to induce Ca2+ transients in almost every cell by blowing a stream of physiological solution onto a single endothelial cell thereby inducing shear stress between 0 and 50 dyn cm-2. The Ca2+ response could be graded by varying the shear stress, and reached a half-maximal value at a shear stress of 30 dyn cm-2. 3. The shear stress responses critically depended on the extracellular Ca2+ concentration and were absent in a Ca(2+)-free solution. Repetitive application of short pulses of shear stress induced cumulative effects because of the slow decay of the shear stress Ca2+ responses (time constants 82.3 +/- 17.8 s from twenty-five cells). Application of a depolarizing high potassium solution to reduce the driving force for Ca2+ entry decreased the Ca2+ transients in some of the cells. 4. Application of shear stress in the presence of other divalent cations, such as nickel, cobalt or barium, always produced substantial changes in the ratio of the 390/360 nm fluorescence signal, indicating influx of these cations and subsequent quenching of the Fura-2 fluorescence. 5. Shear stress responses in the presence of 10 mM Ca2+ were completely blocked by application of 1 mM La3+. 6. Incubation of the cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not alter the shear stress response, but completely blocked histamine-induced Ca2+ transients. 7. Small submaximal shear stress potentiated the Ca2+ transients induced by histamine. 8. We conclude that shear stress-dependent Ca2+ signals are induced by an influx of calcium that is not modulated via protein kinase C and not activated by membrane depolarization. The influx pathway is also permeable to divalent cations such as Ni2+, Co2+ and Ba2+, but is blocked by La3+.


Cell volume changes upon sodium pump inhibition in Helix aspersa neurones. 1. Identified neurones of the suboesophageal ganglia of Helix aspersa were loaded with tetramethylammonium (TMA+). Experimentally induced changes in cell water volume and membrane potential were measured continuously by monitoring changes in intracellular [TMA+] using ion-sensitive double-barrelled microelectrodes. The technique allowed measurements of cell water volume changes of less than 5%. 2. Exposure to hyperosmotic (up to +24%) or hyposmotic (up to about -10%) solutions caused reversible decreases and increases in cell water volume respectively, which agreed with near-ideal osmometric behaviour. Upon exposure to hyposmotic solutions whose osmolality was decreased by 30-40%, the cell water volume increased to maximum values below those expected for ideal osmometric behaviour and exhibited partial regulatory volume decrease. 3. The sodium pump was inhibited in twenty identified neurones by sustained exposure to 1 mM ouabain. In every case ouabain caused cell membrane depolarization, as expected for inhibition of an electrogenic sodium pump. 4. Upon pump inhibition most cells (n = 14) shrank by up to 13% of their initial water volume. In five of these cells, shrinkage was preceded by one or more short-lived swelling phases. In two other neurones short-lived swelling was followed by cell volume recovery without appreciable shrinkage. In four out of the twenty cells, there were no measurable volume changes. 5. The lack of an initial swelling phase in the cells that shrank, as well as the absence of detectable volume changes in some of the neurones, was not due to loss of ion-selective electrode sensitivity since predictable changes in cell volume elicited by osmotic challenges were monitored in the same cells. 6. It is concluded that neurones can be endowed with ouabain-insensitive mechanisms of volume control, whose activation following Na+ pump inhibition prevents them from short-term swelling and lysis.


Substrate influences rat osteoclast morphology and expression of potassium conductances. 1. We studied the electrophysiological properties of freshly isolated rat osteoclasts using the whole-cell configuration of the patch-clamp technique. Membrane currents were recorded from cells plated on three substates: dentine, type I collagen and glass. 2. Based on their morphology, we defined two categories of osteoclasts. 'Rounded' osteoclasts were dome-shaped and lacked lamellipodia. 'Spread' osteoclasts were flattened and had lamellipodia. The proportion of 'rounded' osteoclasts was significantly greater when cells were plated on dentine or type I collagen than when cells were plated on glass. 3. 'Spread' osteoclasts expressed an inwardly rectifying K+ conductance regardless of the substrate on which they were plated. 4. 'Rounded' osteoclasts, on all substrates, expressed a transient, outwardly rectifying conductance that was selective for K+ based on: reversal of deactivation tail currents at -74 mV; a 60 mV shift in tail current reversal potential for 10-fold change in [K+]o; and blockade of outward current by extracellular 4-aminopyridine, charybdotoxin, and intracellular Cs+. The outward K+ current had an activation threshold of approximately -50 mV, with half-activation at -29 mV. The current also exhibited voltage-dependent inactivation, with half-inactivation at approximately -40 mV. 5. Outward K+ current in 'rounded' osteoclasts was reduced when extracellular Ca2+ was removed and upon addition of Ni2+, but was unaffected by Cd2+ or nifedipine. 6. 'Rounded' osteoclasts had large whole-cell capacitance for their apparent surface area. Capacitance was positively correlated with K+ conductance. The additional surface membrane we detected through capacitance measurements may be the 'ruffled border' of actively resorbing osteoclasts. 7. We conclude that substrate influences the expression of osteoclast phenotype, as defined by morphology and K+ conductances. 'Rounded' osteoclasts express an outwardly rectifying K+ conductance, with no apparent inwardly rectifying K+ conductance. In contrast, 'spread' osteoclasts exhibit an inwardly rectifying K+ conductance with no outwardly rectifying K+ conductance. The 'spread' phenotype may represent a motile phase, while the 'rounded' phenotype may represent a resorptive phase of osteoclastic activity.


Role of M1 muscarinic receptors in the parasympathetic control of colonic motility in cats and rabbits. 1. The effects of pirenzepine (a selective blocking agent of M1 muscarinic receptors) were studied on excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) elicited on colonic smooth muscle by stimulation of efferent parasympathetic nerve fibres in anaesthetized cats and rabbits. 2. Pirenzepine (25 micrograms kg-1 to 0.2 mg kg-1, i.v.) decreased the amplitude of EJPs or abolished them. In pirenzepine, parasympathetic stimulation elicited IJPs in most cases. 3. In both species, pirenzepine initiated spontaneous IJPs, indicating an increase in the activity of the non-adrenergic, non-cholinergic (NANC) inhibitory neurones. 4. The results suggest that M1 muscarinic receptors are involved in synaptic transmission within intramural plexuses, at interneuronal synapses in the parasympathetic excitatory pathway to colonic smooth muscle, but are not involved in the pathway to the NANC inhibitory neurones. The facilitation of IJPs by pirenzepine suggests that, under normal physiological conditions, NANC neurones are tonically inhibited by an intramural nervous circuit involving M1 muscarinic receptors.