Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes. Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.


Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells. The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.


GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion. The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.


Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning. The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.


Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone. By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.


A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells. The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to be derived from the progenitor double-positive T cells. These results suggest the existence of similar and common factors in CD4+ CD8- and CD4- CD8+ T cells and support a model of differentiation of CD4+ CD8+ T cells through common signal(s) involved in turning off the expression of the CD4 or CD8 gene.


Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.


pp39mos is associated with p34cdc2 kinase in c-mosxe-transformed NIH 3T3 cells. We investigated the possible interactions between pp39mos and p34cdc2 kinase in NIH 3T3 cells transformed by c-mosxe. pp39mos is coprecipitated with p34cdc2 when using either anti-PSTAIR antibody or p13suc1-Sepharose beads. Likewise, p34cdc2 is coprecipitated with pp39mos when using anti-mos antibody. However, pp39mos was not present in histone H1 kinase-active p34cdc2 complexes precipitated with anti-p34cdc2 C-terminal peptide antibody even during metaphase of the cell cycle. The molar ratio of p34 to pp39mos in the p13suc1 complex is approximately 2:1. Consistent with the tight association between pp39mos and tubulin, tubulin was also present in equivalent amounts with pp39mos and p34 in the p13suc1 complex. This pp39mos-p34cdc2-tubulin complex may be important in transformation by the mos oncogene.


Different roles for two enhancer domains in the organ- and age-specific pattern of polyomavirus replication in the mouse. Viral replication in mice infected with murine polyomavirus strains with novel enhancer rearrangements was analyzed by direct in situ hybridization of whole mouse sections and by hybridization of nucleic acids extracted from a specific set of organs. The enhancer rearrangements included a deletion of the B domain as well as duplications within the A domain. Comparisons between enhancer variants demonstrate that the B domain plays an important role in replication in most organs, in particular in the kidney, at the neonatal stage (days 0 to 7 postbirth). In contrast, the B domain is not required in those organs which can sustain replication in the adult, i.e. mammary gland, skin, and bone (class I organs [J. J. Wirth, A. Amalfitano, R. Gross, M. B. A. Oldstone, and M. M. Fluck, J. Virol. 66:3278-3286, 1992]). Altogether, the results suggest that the B and A domains mediate very different functions in infection of mice, controlling the acute and persistent phases of infection, respectively. A model of mouse infection based on the crucial role of differentially expressed host transcription factors is presented.


Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus. Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.


Structure, expression, and chromosome location of the gene for the beta subunit of brain-specific Ca2+/calmodulin-dependent protein kinase II identified by transgene integration in an embryonic lethal mouse mutant. The transgenic mouse strain CAT40 carries in its germ line one copy of a DNA construct consisting of the chloramphenicol acetyltransferase gene and the immunoglobulin heavy-chain enhancer. We show that transgene integration has resulted in a recessive lethal mutation that leads to death of homozygous CAT40 embryos shortly after implantation. The transgene has integrated adjacent to the 3' end of the gene coding for the beta subunit of the brain-specific Ca2+/calmodulin-dependent protein kinase II (Camk-2). The complete cDNA sequence of the Camk-2 gene and most of its exon/intron structure was determined. The deduced amino acid sequence is highly homologous to the previously described rat protein. The chromosomal location of the Camk-2 locus was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 11. This region lacks previously identified recessive embryonic lethal mutations. During embryonic development, Camk-2-specific transcripts are first seen in the head section of 12.5-day-old embryos, and in adult mice the gene is expressed almost exclusively in the brain. Although transcription of the Camk-2 gene in heterozygous CAT40 mice is affected by transgene integration, it is unlikely that this gene is responsible for the mutant phenotype, since it is not expressed in blastocysts and the first transcripts during normal development are detected after the death of homozygous CAT40 embryos. Transgene integration is accompanied by a large deletion of cellular DNA; death is therefore most likely caused by the loss of a gene or genes that are important for early postimplantation development.


Positive and negative elements regulate a melanocyte-specific promoter. Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis- and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-1-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed.


The Lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase. Lowe's oculocerebrorenal syndrome (OCRL) is a human X-linked developmental disorder of unknown pathogenesis and has a pleiotropic phenotype affecting the lens, brain and kidneys. The OCRL locus has been mapped to Xq25-q26 by linkage and by finding de novo X; autosome translocations at Xq25-q26 in two unrelated females with OCRL. Here we use yeast artificial chromosomes with inserts that span the X chromosomal breakpoint from a female OCRL patient in order to isolate complementary DNAs for a gene that is interrupted by the translocation. We show that the transcript is absent in both female OCRL patients with X; autosome translocations and that it is absent or abnormally sized in 9 of 13 unrelated male OCRL patients with no detectable genomic rearrangement. The open reading frame encodes a new protein with 71% similarity to human inositol polyphosphate-5-phosphatase. Our results suggest that OCRL may be an inborn error of inositol phosphate metabolism.


Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2 and phosphatidylinositol 4-phosphate (PtdInsP) in vitro and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are cultured in the presence of insulin-like growth factor-1 (IGF-1). These IGF-1-dependent changes in inositol lipids coincide with an increase in nuclear diacyglycerol and precede translocation to the nucleus and activation of protein kinase C (refs 5, 6). Circumstantial evidence that links these changes with mitosis comes from the isolation of a 3T3 clone that expresses the type-1 IGF receptor and binds IGF-1 peptide but does not respond mitogenically or show transient mass changes in nuclear inositol lipids. A key question is how IGF-1 initiates the rapid breakdown of PtdInsP and PtdInsP2 in the nucleus. Here we present evidence that nuclei of 3T3 cells contain the beta-isozyme of phosphoinositidase C, whereas the gamma-isozyme is confined to the cytoplasm and that IGF-1 treatment stimulates exclusively the activity of nuclear phosphoinositidase C.


Retinoblastoma protein switches the E2F site from positive to negative element. Originally E2F sites were identified as elements in the promoters of adenovirus early genes that are necessary for activation of these genes by the early protein E1a (ref. 1). E2F promoter elements have been shown to be important for transcriptional activation of several genes critical for progression through the cell cycle. During the G1 phase of the cell cycle, the E2F protein forms a complex with the cell-cycle protein Rb (ref. 5) and it has been suggested that this binding of Rb to E2F inactivates E2F (ref. 5). Here we show that Rb-E2F is an active complex that, when bound to the E2F site, inhibits the activity of other promoter elements and thus silences transcription. We propose that the ability of this complex to inhibit transcription is integral to the function of Rb and provide evidence that E2F is a positive element in the absence of an active form of Rb. It has been shown that binding of Rb to E2F depends on the phosphorylation state of Rb (only the underphosphorylated form binds) and that the phosphorylation state of Rb changes during progression through the cell cycle. We therefore suggest that the E2F site alternates between a positive and negative element with the phosphorylation/dephosphorylation cycle of Rb. This cyclic activity may be responsible for activating and then inhibiting genes during the cell cycle.


Minimal change nephrotic syndrome revealing solid tumors. In 2 patients with the nephrotic syndrome, unsuspected solid tumors were found. One was a small cell lung carcinoma, accompanied with the syndrome of inappropriate ADH secretion. The other was a cancer of the breast with lymph node and bone metastases. In both, renal biopsy showed minimal change disease without immune complex deposits. There are only 14 other reported cases of paraneoplastic lipoid nephrosis complicating solid tumors. Such cases lead to the discussion on the respective roles of tumor cell gene product(s) inducing proteinuria and of lymphokine secretion by lymphocytes directed against the tumor itself. Cancer should be considered as a possible etiology of the minimal change nephrotic syndrome in the adult.


[Sphenoid sinus aspergillosis presenting abducens nerve palsy and visual field impairment; a case report]. A case of sphenoid sinus aspergillosis presenting abducens nerve palsy and visual field impairment is reported. A 73-year-old woman visited our hospital with the complaint of head heaviness on the 27th of March, 1989. Although results of neurological examinations were normal, craniogram revealed the destruction of the clivus, and CT scan and MRI showed a mass lesion, which was thought to be a mucocele in the sphenoid sinus. On the 1st of September, she developed right abducens nerve palsy and visual field impairment. MRI performed on the same day showed an enlargement of the mass lesion in the sphenoid sinus. In order to decompress the involved cranial nerves, her sphenoid sinus was explored on the 22nd of September. The sphenoid sinus was filled with purulent fluid and yellowish mass. Histopathological examination revealed colonies of aspergillus fumigatus. Fluconazole, a new antifungal drug, was given for 34 days postoperatively. The right abducens nerve palsy and the visual field impairment gradually improved along with a reduction of the mass lesion in her sphenoid sinus. Sphenoid sinus aspergillosis is a rare disease. Its diagnosis is difficult. However, MRI can show a specific low signal intensity in T2-weighted image. Also in our case, MRI on the first admission showed a definite low signal intensity in some parts of the lesion, which exhibited a high intensity later on during the second administration, probably due to a qualitative change. To our knowledge, only 33 such cases have been previously reported. Intracranial involvement occasionally occurs in this disease. In its early stage, cranial nerve palsies are caused by nerve compression or invasion by this disease.(ABSTRACT TRUNCATED AT 250 WORDS)


[Reconstruction of anterior cranial base and face with free musculocutaneous flap; report of two cases]. The treatment of extensive cranial base tumor is still challenging. Wide resection of a cranial base tumor would lead to exposure of the vital structures such as the brain and dura, and would result in a large dead space in the cranium and in the connecting area between the nasal cavity and the intracranial space. Recently free tissue transfer has become a reliable method in the field of reconstructive surgery. In the craniofacial region, free tissue transfer is useful to reconstruct a complicated defect. We report two cases where free latissimus dorsi M-C flap and rectus abdominis M-C flap were useful in reconstructing the defect following removal of an anterior cranial base tumor. We discuss the advantages.


Immunocytochemical localization of ubiquitin at human neuromuscular junctions. Using several specific monoclonal and polyclonal antibodies, we found that ubiquitin is highly concentrated in the post-synaptic domain of human neuromuscular junctions. The distribution and pattern of ubiquitin immunoreactivity is identical to the localization of bound alpha-bungarotoxin, suggesting that ubiquitin may be involved in post-translational modification or turnover of the acetylcholine receptor or other junctional proteins of the muscle fibre.


Antidepressants increase glucocorticoid and mineralocorticoid receptor mRNA expression in rat hippocampus in vivo. Adrenal corticosteroids bind to hippocampal glucocorticoid (GR) and mineralocorticoid receptors (MR), thereby affecting neurochemical transmission leading to altered mood, behaviour and neuroendocrine control. Serotoninergic (5-HT) and noradrenergic projections innervate the hippocampus, interacting with corticosteroid-sensitive cells. We have previously demonstrated that lesions of 5-HT neurons reduce hippocampal GR and MR mRNA levels and now examine whether acute or chronic treatment with antidepressant drugs, which potentiate endogenous monoamines by inhibiting their reuptake, affect hippocampal GR and MR mRNA expression in vivo. Rats were treated with amitriptyline (20 mg/kg.day-1), desipramine (10 mg/kg.day-1) or citalopram (20 mg/kg.day-1). After 2 or 14 days animals were killed, RNA extracted and GR and MR mRNA expression quantified by slot blot hybridization. Amitriptyline for 2 days led to a significant increase in MR (by 23 +/- 6%, compared with saline-treated controls), but not GR, mRNA expression. After 14 days amitriptyline, expression of both MR (87 +/- 27% rise) and GR mRNA (56 +/- 18% rise) had increased significantly in hippocampus, but not in parietal cortex. Desipramine for 14 days also increased MR (60 +/- 9%) and GR (42 +/- 9%) mRNA expression, though 14 days of citalopram altered only MR mRNA expression (17 +/- 5%). Thus, antidepressant drug administration elevates MR and GR mRNA expression in hippocampus, but not parietal cortex, in a time-dependent manner.


Interleukin-1-beta induces pituitary adrenocorticotropin secretion: evidence for glucocorticoid modulation. Using an in vitro continuous perifusion system, the effects of interleukin-1 (IL-1) on adrenocorticotropin (ACTH) secretion at the pituitary level were investigated. On one hand, we observed that IL-1 beta increases ACTH secretion from perifused anterior pituitary cells in a dose-dependent manner, between 1.5 and 6 pM. This stimulatory action of IL-1 on ACTH was significantly attenuated by a short in vitro dexamethasone pretreatment. This fact suggests a regulatory glucocorticoid negative feedback analogous to that observed upon the pituitary action of corticotropin-releasing factor (CRF). We also examined the effect of simultaneous treatment with IL-1 and CRF. The results indicated that the effect of IL-1 resulted additive to the CRF-induced ACTH secretion. It is concluded that the anterior pituitary could be an important site of IL-1 action to activate the hypothalamic-pituitary-adrenocortical axis function, and that this action is under an inhibitory glucocorticoid regulation.


Hypoglycemia-induced arginine vasopressin and oxytocin release is mediated by glucoreceptors located inside the blood-brain barrier. Arginine vasopressin (AVP) and oxytocin (OT) responses during an insulin (0.15 IU/kg body weight) tolerance test (ITT) were evaluated in normal men while they were infused with normal saline, glucose or fructose. Insulin-induced hypoglycemia produced significant plasma AVP and OT increments in the control test. The infusion of fructose was unable to change the posterior pituitary hormonal responses to hypoglycemia. In contrast, AVP and OT responses during ITT were completely abolished when the concomitant infusion of glucose prevented insulin-induced hypoglycemia. These data exclude a direct role of hyperinsulinemia in the mechanism underlying the AVP and OT responses during ITT. Furthermore, since glucose, but not fructose, crosses the blood-brain barrier (BBB), the posterior pituitary hormone responses to hypoglycemia appear to be generated by stimulations of glucosensitive areas located inside the BBB.


Steroid modulation of the strychnine-sensitive glycine receptor. Electrophysiological responses to glycine (0.25-5 mM) were obtained on preparations of rat optic nerve. The log dose-response curve for glycine was shifted to the left by a factor of 2 by 1 microM 20 alpha-dihydrocortisol and by a factor of about 1.5 by 1 microM alpha-cortol and 10 microM hydrocortisone. A similar effect was obtained with 100 microM chlormethiazole but the GABA-potentiating steroid alphaxalone (1 microM) was ineffective on responses to glycine. 20 alpha-Dihydrocortisol and chlormethiazole also appeared to increase the antagonistic potency of strychnine against glycine. These observations suggest that the active steroids and chlormethiazole increase the functional interaction of both glycine and strychnine with the glycine-gated chloride channel.


Characteristics of endothelin receptors in the central nervous system of spontaneously hypertensive rats. The binding of [125I]sarafotoxin 6b (SRT 6b) and [125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared, from regions of the brain and spinal cord of 8 week-old, spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. Spontaneously hypertensive rats had significantly higher blood pressure as compared to WKY rats. Heart rate was similar in SHR and WKY rats. [125I]SRT 6b and [125I]ET-1 bound to the membranes of the cerebral cortex, hypothalamus, ventrolateral medulla, dorsomedial medulla and spinal cord at a single, high affinity site. The Kd and Bmax values of the binding of [125I]SRT 6b were found to be similar to binding of [125I]ET-1 in all the regions. The concentration-dependent inhibition of binding of [125I]ET-1 by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 2.66 +/- 0.59 nM and a Ki value of 2.35 +/- 0.52 nM in WKY rats and an IC50 value of 2.82 +/- 0.76 nM and a Ki value of 2.43 +/- 0.70 nM in SHR rats. On the other hand, the concentration-dependent inhibition of the binding of [125I]SRT 6b by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 10.31 +/- 1.82 pM and a Ki value of 8.44 +/- 1.41 pM in WKY rats, while SHR rats showed an IC50 value of 10.28 +/- 1.94 pM and a Ki value of 8.89 +/- 2.00 pM. The binding of [125I]SRT 6b and [125I]ET-1 in the cerebral cortex, dorsomedial medulla and spinal cord membranes was found to be similar in SHR and WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS) [corrected]


Degenerative and axon outgrowth-altering effects of phencyclidine in human fetal cerebral cortical cells. Babies born to mothers abusing the psychotomimetic drug phencyclidine (PCP), often show profound deficits in CNS function. It is now reported that PCP can cause progressive degeneration and death in human fetal cerebral cortical neurons in culture and sublethal levels of PCP can inhibit axon outgrowth. In cerebral cortical cell cultures established from 14 week fetuses, exposure to 500 microM PCP resulted in a progressive degeneration of neurons over a 2-8 day period, characterized by early reversible vacuolation of the soma and later irreversible neurite fragmentation and cell death. A sublethal concentration of PCP (100 microM) suppressed axon outgrowth. The adverse effects of PCP on neurons were apparently not due to actions on N-methyl-D-aspartate (NMDA) receptors because: the neurons were not responsive to NMDA during the first 3 weeks in culture; concentrations of PCP that should block NMDA receptors maximally (1-50 microM) did not affect axon outgrowth or cell survival; and another NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) did not cause neurodegeneration or affect axon outgrowth. Exposure of cultures to the sigma receptor ligand, pentazocine (10 microM), did not significantly affect the survival of neurons and haloperidol did not reduce PCP-induced neurodegeneration, indicating that the effects of PCP were not mediated by sigma receptors. Degenerative effects, similar to those elicited by PCP, were observed in cortical neurons exposed to the K+ channel blockers, 4-aminopyridine and tetraethylammonium, a finding consistent with the possibility that the degenerative actions of PCP were mediated by its known inhibitory effects on K+ channels. In support of the latter possibility, it was found that PCP caused a progressive elevation in intracellular levels of calcium during several days of exposure. In addition to affecting neurons, PCP and K+ channel blockers caused vacuolation and degeneration of astrocytes. Taken together with previous data, indicating that PCP can be concentrated and retained in the fetal CNS, the present data suggest that high levels of PCP can disrupt the normal development of neural circuitry in the human fetus, which would be expected to result in profound functional impairments.


Inhibition of enkephalin metabolism and activation of mu- or delta-opioid receptors elicit opposite effects on reward and motility in the ventral mesencephalon. The coexistence of endogenous opioid systems and dopaminergic neurones in the midbrain tegmental area suggests functional interactions between dopamine and enkephalins. Nevertheless, the identification of the specific opioid receptors associated with modulation of tegmental dopamine activity and its behavioural concomitants on motility and reward is far from clear, considering the mixed nature of the ligands usually employed. In this way, kelatorphan, a potent inhibitor of enkephalinases and selective agonists for mu- and delta-opioid receptor subtypes (DAGO and DSTBULET, respectively) were infused directly into the ventral tegmental area of the rat to study the role of endogenous enkephalins and opioid receptors in regulating spontaneous motor activity and intracranial self-stimulation behaviour. A greater increase in the rate of intracranial self-stimulation behaviour was found after activation of mu-opioid receptors in the ventral tegmental area, as compared to activation of delta-opioid receptors, whereas enhancement of endogenous enkephalins by inhibiting their metabolism through kelatorphan, reduced the rate of intracranial self-stimulation behaviour. On the contrary, spontaneous motor activity was reduced by the delta-opioid receptor agonist, whereas kelatorphan increased the movements of the animal. Taken together, these results show that inhibition of the metabolism of enkephalins in the ventral tegmental area decreased positive reinforcement from the lateral hypothalamic medial forebrain bundle and increased spontaneous movements. On the contrary, activation of both mu- or delta-opioid receptors in the ventral tegmental area significantly increased self-stimulation and decreased spontaneous motor activity, supporting the view that different mechanisms underlie the behavioural effects, resulting from enhancement of endogenous enkephalins and from activation of specific opioid receptors in the ventral mesencephalon.


Pharmacological heterogeneity among calcium channels that subserve acetylcholine release in vertebrate forebrain. Inhibition of calcium-evoked [3H]ACh release by different classes of calcium channel blockers was compared among calcium-naive synaptosomes from chick, frog and rat forebrain tissues. In all three species, [3H]ACh release was insensitive to nifedipine (0.03-3 microM) but was completely inhibited by cadmium (IC50 range = 0.7-1.7 microM) or cobalt (190-350 microM). In contrast, the peptide omega-conotoxin revealed marked species heterogeneity in that [3H]ACh release was potently blocked in chick and rat synaptosomes (IC50 congruent to 1 nM), whereas frog tissue was notably resistant (IC50 greater than 100 nM). Together, these data provide functional evidence for pharmacological heterogeneity among presynaptic calcium channels that subserve [3H]ACh release.


Intrathecal cholecystokinin octapeptide attenuates the antinociception and release of immunoreactive Met-enkephalin induced by intraventricular beta-endorphin in the rat. The effects of cholecystokinin octapeptide (CCK8s) given intrathecally (i.t.) on antinociception and the release of immunoreactive Met-enkephalin in the spinal perfusate induced by intraventricular (i.vt.) injection of beta-endorphin were studied in anesthetized rats. beta-Endorphin (5 micrograms) given i.vt. inhibited the tail-flick response. The inhibition of the tail-flick response induced by beta-endorphin was blocked dose-dependently by CCK8s (0.1-7 micrograms) given i.t. The antagonistic effect of CCK8s on beta-endorphin-induced inhibition was blocked dose dependently by co-intrathecal injection of proglumide (3 and 10 micrograms), a CCK8s receptor antagonist. beta-Endorphin (5 micrograms) given i.vt. elicited a release of immunoreactive Met-enkephalin in the spinal perfusate. Repeated injections of the same dose of beta-endorphin released about the same amount of the immunoreactive Met-enkephalin in the spinal perfusate. CCK8s at concentrations from 1 x 10(-9) to 1 x 10(-6) M added into the spinal perfusate decreased the release of Met-enkephalin induced by beta-endorphin given i.vt. in a dose-dependent manner. The results suggest that CCK8s may attenuate beta-endorphin-induced inhibition of the tail-flick response by inhibiting the release of Met-enkephalin from the spinal cord.


Calcitonin gene-related peptide depresses the growth and secretory activity of rat adrenal zona glomerulosa. The bolus ip. injection of rat calcitonin gene-related peptide (CGRP) (5 pm. kg-1) significantly lowered plasma aldosterone concentration (PAC) in rats, despite a mild rise in plasma renin activity. Natremia, kalaemia and the blood levels of ACTH or corticosterone were not affected. Similar results were obtained after prolonged (5 days) sc. infusion of rats with CGRP (1 pm. kg-1. h-1). Moreover, CGRP infusion caused a notable atrophy of the zona glomerulosa (ZG) and its parenchymal cells, as well as a clearcut reduction in the surge of PAC evoked by a bolus injection of a high dose of angiotensin-II (100 micrograms. kg-1). From these results it is suggested that CGRP exerts an inhibitory effect on the growth and secretory activity of ZG in rats.


Ex vivo determination of opiate antagonist binding at mu-opioid ([3H]-DAGO) receptors in hypothalamic micropunches from maturing female rats: comparison between SDZ 210-096 and nalmefene. We have previously shown that the duration of opioid receptor blockade is critical in determining the degree of opioid antagonist effect following peripheral injection of naloxone and naltrexone. In the present work, we have used this ex vivo technique to compare receptor occupancy of a new opiate antagonist, SDZ 210-096 (SDZ), to that of nalmefene (NLM) in maturing female rats. Two doses (SDZ, 5.6 and 50 mg/kg; NLM, 2.5 and 50 mg/kg) were injected subcutaneously into 3 groups of rats (infantile, juvenile and peripubertal). Micropunches from hypothalamic coronal slices (300 microns) were removed at various times post-injection for quantification of mu-opioid receptors with [3H]-DAGO. Acute administration of the lower dose of SDZ inhibited ligand binding almost completely by 3 h but 50% recovery was observed in all age groups by 12 h. In contrast, SDZ 50 mg/kg provided 80-100% antagonism for at least 24 h. Age-related differences in the ability of SDZ to inhibit [3H]-DAGO binding were observed in that hypothalamic mu-opioid receptors were blocked for longer periods in younger rats. Determination of receptor occupation following NLM injection confirmed that it too has prolonged duration of action but a 24 h blockade is not achieved with either dose of this antagonist. Age-related and dose-related changes in receptor occupancy were minimal compared to SDZ. These studies clarify the interaction of these antagonists at hypothalamic mu-opioid receptors and provide information which allows a clearer interpretation of results in experiments involving opioid blockade.


Effects of central and peripheral type benzodiazepine ligands on thyrotropin and prolactin secretion. The cold-stimulated thyrotropin (TSH) levels in the rat were decreased by clonazepam (a central type benzodiazepine agonist), diazepam (a mixed agonist), FG 7142 (an inverse central type agonist) and Ro 5-4864 (a peripheral type agonist), clonazepam being the most potent and Ro 5-4864 the least active. Clonazepam and diazepam also decreased while FG 7142 increased prolactin (PRL) levels. Ro 5-4864 did not have any significant action. Clonazepam (1 and 5 mg/kg) and diazepam (15 mg/kg but not 25 mg/kg) decreased even the TRH-induced PRL levels. Only Ro 5-4864 (25 mg/kg) decreased TRH-induced TSH secretion but not significantly. The actions of central type compounds were antagonized by flumazenil but not by PK 11195. The weak effects of Ro 5-4864 were not antagonized by either antagonists. While the peripheral type benzodiazepine agonist only weakly affected the secretion of anterior pituitary hormones, the central type inhibition of TSH appears to be mediated through the hypothalamic TRH and that of PRL rather through the anterior pituitary gland. The sedating (or agitating in case of FG 7142) effect of high doses of benzodiazepine ligands may contribute to the changes in TSH and PRL levels.


Vitamin D nuclear binding to neurons of the septal, substriatal and amygdaloid area in the Siberian hamster (Phodopus sungorus) brain. Autoradiographic experiments were performed on brains of Siberian hamsters (Phodopus sungorus) injected with tritiated 1,25-dihydroxycholecalciferol. Nuclear labeling was prevented in the presence of excess unlabeled hormone. Strong nuclear concentration of radioactivity was observed in neurons of the nucleus basalis of Meynert, the medial septal nucleus, the nucleus of the diagonal band of Broca and the central amygdaloid group. The latter has been defined as consisting of the central nucleus of the amygdala, its extension into the sublenticular part of the substantia innominata of Reichert, and the lateral division of the bed nucleus of the stria terminalis. All these structures have been reported to be involved in memory and other cognitive processes, and to be affected by age-dependent neurodegenerative disorders such as Alzheimer's disease. Corresponding localization of 1,25-dihydroxycholecalciferol receptor sites in these select basal forebrain nuclei of the Siberian hamster may implicate vitamin D (soltriol), the steroid hormone of sunlight, in memory processing.


Regional distribution of guanine nucleotide-sensitive and guanine nucleotide-insensitive vasoactive intestinal peptide receptors in rat brain. Based on the ability of guanine nucleotides to inhibit the binding of vasoactive intestinal peptide to its receptors, a guanosine 5'-triphosphate analog, guanylyl-imidodiphosphate, was used to differentiate two subtypes (or different functional states of a single subtype) of vasoactive intestinal peptide receptor in brain with in vitro autoradiography. In most brain regions, guanylyl-imidodiphosphate reduced vasoactive intestinal peptide binding between 40 and 60%. However, in the supraoptic nucleus, locus coeruleus, interpeduncular nucleus, facial nucleus, olfactory tubercle and periventricular hypothalamic nucleus, 80% or more of vasoactive intestinal peptide binding was inhibited. In other brain regions, including the medial geniculate, olfactory bulbs, and ventral thalamic nuclei, guanylyl-imidodiphosphate had little effect on vasoactive intestinal peptide binding. In liver, lung and intestine it also partly inhibited vasoactive intestinal peptide binding. Electrophoretic analysis of vasoactive intestinal peptide, covalently cross-linked to its receptors in brain membranes, revealed a pair of bands between 44,000 and 52,000 mol. wt, a component at 64,000 mol. wt and another at 92,000 mol. wt. All were displaceable with vasoactive intestinal peptide but guanylyl-imidodiphosphate displaced only the 64,000 mol. wt band suggesting that the GTP-sensitive vasoactive intestinal peptide receptor seen in brain sections has a molecular weight of about 61,000. The differential sensitivity to guanylyl-imidodiphosphate suggests the existence of at least two vasoactive intestinal peptide receptor subtypes in brain, with distinct regional distribution, and may reflect differential coupling to second messenger systems.


[Rare neoplasms of the pancreas: the papillary-cystic tumor. A report of 2 cases]. The authors report two cases of papillary-cystic tumor of the pancreas in two young women. Making a review of literature, they emphasize the rarity of this neoplasm and analyze the possible histopathologic origin, the difficult diagnosis before surgery, surgical therapy and the good prognosis of this pancreatic "curable" tumor.


[Radiation-surgical treatment of cancer of the female breast]. Findings were reviewed of 1566 female patients with primary breast carcinoma treated with radical or total mastectomy followed by postoperative radiation therapy during the 22 year period 1960-82. The five-year overall survival for the axillary node-positive breast cancer group was 58.3 percent, as compared with 79.9 percent for the node-negative group. The survival rates were also analyzed in terms of age of the patients and number of the axillary positive nodes present. The ratios of observed deaths to expected deaths were turned from many times greater than unity into nearly the expected number, after ten year follow-up period. These findings showed that host factors are capable of destroying tumor cells both in node-negative and in node-positive patients. The significance of axillary negative nodes was emphasized. The rates of locoregional recurrences and distant metastases for node-negative patients were analyzed; these observations suggested that routine postoperative radiotherapy should be considered also in node-negative breast cancer patients.


Circumferential mesangial interposition: a form of mesangiolysis. A wide variety of glomerular lesions express circumferential mesangial interposition (CMI). The pathomorphogenesis of CMI involves low-grade mesangiolysis and subsequent passive dislocation of mesangial cells towards the lateral wall of glomerular capillaries through a high hydraulic pressure of blood flow penetrating the lytic mesangium. This is in contrast to the previous theories which advocate an active movement of mesangial cells. Moreover, circumferential mesangial interposition can disappear spontaneously or after treatment, suggesting that mesangial cells may return to the original site with their inherent contractility after the insults are removed, in order to restore handicapped filtering surfaces. But the precise mechanisms of its regression will be a subject for future investigation.


Natural history of chronic renal failure: a reappraisal. In this retrospective study we have analysed the rate of progression of renal insufficiency, ascertained from the slopes of the plot of inverse serum creatinine against time, of 102 patients with moderate to severe chronic renal failure (CRF). We have applied 'breakpoint' analysis of the slopes to identify changes in the rate of progression and attempted to determine the factors associated with these changes. Seventy-one patients were found to have progressive CRF, while the remaining 31 had stable or improving renal function. Of the parameters studied, using weighted least-squares analysis, proteinuria was the most significant predictor of progression (regression coefficient: -0.1775, P = 0.0075, adjusted r2 = 0.1059). A positive correlation was observed between proteinuria and diastolic blood pressure (DBP) (r = 0.336, P = 0.0054). Once the predictive value of proteinuria was taken into account, there was no difference in the progression rate between diagnostic groups, other than those patients with polycystic kidney disease who had a significantly faster rate of progression (P = 0.0037). In 49 patients, there was at least one change in the rate of progression with time. There was an inverse correlation between change in slope and a change in DBP (r = -0.352, P = 0.003). We conclude that changes in DBP are often associated with the frequent changes in the rate of progression of CRF. However, a causal link could not be established as in a large number of cases the two changes appeared to occur simultaneously in the absence of changes in antihypertensive therapy.


Pattern of renal dysfunction in analgesic nephropathy--comparison with glomerulonephritis. Comprehensive renal function tests were performed in 84 patients with analgesic nephropathy, 33 glomerulonephritis patients matched for creatinine clearance, and 30 control subjects. A system of 1-day renal function tests including urine microscopy, creatinine clearance, phenolsulphonphthalein excretion, urine concentration and acidification, and electrolyte excretion, was used. Patients with analgesic nephropathy were found to have significant sterile pyuria and haematuria, even those with mild renal insufficiency, significantly reduced concentrating ability and a distal acidifying defect, and a tendency to impaired sodium conservation. These function defects are consistent with the primary lesion of renal papillary necrosis in analgesic nephropathy; the detection of these defects have implications in patient management.


C1q nephropathy: do C1q deposits have any prognostic significance in the nephrotic syndrome? C1q deposits are usually found in association with other complement components and immunoglobulins in proliferative glomerulonephritis and may predominate in systemic lupus erythematosus (SLE). We report the clinical outcome of four patients who developed a nephrotic syndrome associated with C1q nephropathy unrelated to SLE. On presentation the mean urinary protein loss was 6.8 g/24 h (range 4-10), and renal function impaired, mean serum creatinine 201 mumol/l (150-400). Over a mean follow up period of 6.5 years (1.7-19), all four patients improved, three spontaneously and one treated with steroids and cyclosporin, to a current urinary protein loss of 0.3 g/24 h (less than 0.2-0.9) and serum creatinine 98 mumol/l (68-115). C1q nephropathy was confirmed in each biopsy by conventional immunohistology. C1q deposits were demonstrated within the glomerular basement membrane of three biopsies and the mesangium in two samples. One patient had been categorized on light- and electron-microscopy as having mesangiocapillary glomerulonephritis, one membranous glomerulonephritis, one proliferative glomerulonephritis with focal segmental glomerulosclerosis, and one diffuse proliferative glomerulonephritis with both subendothelial and mesangial dense deposits. In view of the expected progressive nature of the underlying renal histopathological appearance, the presence of predominant C1q deposits would appear to be associated with a better clinical outcome.


Increased prevalence of renal biopsy findings other than diabetic glomerulopathy in type II diabetes mellitus. It is a widely held view that when a patient with type I diabetes mellitus and diabetic retinopathy or neuropathy develops renal impairment the renal lesion will be diabetic glomerulonephropathy. This has been extrapolated to apply to type II diabetes. We have performed a retrospective study of the clinical data of patients with diabetes mellitus who have had a renal biopsy between November 1980 and December 1990. Seventy-one patients were biopsied, data were available on 68. Nineteen of 22 type I diabetics had diabetic glomerulopathy, two had diabetic glomerulopathy in addition to another lesion only one patient did not have diabetic glomerulopathy. Twenty-three of 46 type II diabetics had diabetic glomerulopathy alone 22 having an alternative diagnosis. Eight further patients were identified who were not known to be diabetic at the time of renal biopsy, but whose biopsies revealed diabetic glomerulopathy. These data suggest that patients with type II diabetes and renal impairment should have a renal biopsy as part of their investigation.


Diagnostic use of renal vein erythropoietin measurements in patients with renal artery stenosis. The relationship between the renin-angiotensin system and erythropoietin was studied in twenty patients with renal artery stenosis and hypertension. Ten of the patients had a unilaterally activated renin-angiotensin system (group 1), while ten patients had not (group 2). Plasma erythropoietin was simultaneously measured in a brachial artery and both renal veins before and 5 and 30 min after an intravenous injection of 1.25 mg enalaprilat. The mean (+/- SD) arterial erythropoietin concentration was 27.3 +/- 16.8 mU/ml in group 1 and 14.1 +/- 11.3 mU/ml in group 2 patients (P less than 0.05). There was no significant change after enalaprilat i.v. in either group. The venous erythropoietin concentration in plasma from the stenotic kidney did not differ from that of the contralateral kidney. The higher erythropoietin concentration in group 1 patients may be explained by a systemic stimulatory effect of the renin-angiotensin system on erythropoietin production. As no side-differences were found, renal vein as well as peripheral erythropoietin measurements cannot be used as a tool in the diagnosis of the functional significance of a renal artery stenosis.


Minor degree of reversible renal insufficiency: a frequent complication of adult minimal-change nephrotic syndrome. We report on a group of 27 patients with minimal-change nephrotic syndrome who presented complete recovery from the nephrotic syndrome and the other clinical signs during follow-up. At the start of the examination an elevated serum urea was found in 10 (37%) and of serum creatinine in 4 (15%) patients. Creatinine clearance was decreased in 10 (37%), systolic blood pressure was elevated in 8 (29%) and diastolic in 11 (41%). Interstitial oedema was found in 3, tubular parenchymal degeneration in 4, slight interstitial mononuclear infiltration in 6 and slight interstitial fibrosis in 1 patient. Complete recovery of renal function was seen in all patients.


Ultrasound-guided percutaneous fine-needle ethanol injection into parathyroid glands in secondary hyperparathyroidism. To reduce parathyroid hormone concentrations in uraemic patients refractory or hyporesponsive to calcium supplements and active metabolites of vitamin D, we developed in 1982 a new parathyroid ablative technique consisting of percutaneous fine-needle ethanol injection (PFNEI) into enlarged parathyroid glands under ultrasonic guidance. Fifty uraemic patients have been treated. Decreases in carboxy terminal parathyroid hormone (PTH) were 50% or more in 13 of 50 patients followed up (26%) at 1 month, in 13 of 48 (27%) at 6 months, and in 9 of 25 (36%) at 12 months. Decreases of 30% or more in PTH were obtained in 21 of 50 (42%), in 25 of 48 (52%), and in 15 of 25 (60%). In 'responsive' patients, serum total alkaline phosphatase was significantly reduced [from 579 +/- 645 U/l to 360 +/- 354 U/l (P less than 0.01) at 6 months, and to 273 +/- 311 U/l (P less than 0.01) at 12 months] and bone isoenzyme decreased similarly [from 482 +/- 608 U/l to 256 +/- 344 U/l (P less than 0.005), and to 225 +/- 354 U/l (P less than 0.01)]. The best results were in seven patients who had relapsed after subtotal parathyroidectomy. Declines in PTH of 30% or more were observed in four of seven patients at 1 month, in six of the seven (85%) at 6 months, and in all four patients seen after 12 months. The treatment corrected hypercalcaemia, making it possible to start or to increase daily vitamin D treatment. Side-effects were mild, rare, and transient.


Anti-lymphocyte, anti-monocyte, and anti-endothelial cell antibodies in chronic haemodialysis patients. Patients receiving chronic haemodialysis treatment are known to have a high prevalence of anti-panel antibodies (anti-lymphocyte, anti-monocyte, and anti-endothelial cell) originating from a number of different possible sensitizing events such as blood transfusions, multiparity, or renal transplantation. In this study the prevalence of these antibodies in 123 chronic haemodialysis patients was evaluated. Anti-lymphocyte antibodies and anti-monocyte antibodies were found in 27.6% and 21.9% of the patient group respectively. Following exclusion of twelve patients within the study population with a history of previous graft rejection, anti-monocyte antibodies were encountered more frequently in transfused patients than those never having been transfused (22.4% versus 0%, P less than 0.05), whilst the prevalence of anti-lymphocyte antibodies was similar in both groups (28.2% versus 19.2%). Multiparity, patient age, or duration of dialysis treatment could not be demonstrated to significantly alter the prevalence of either of these antibodies. Of 12 patients with an episode of previous graft rejection, eight were positive for anti-monocyte antibodies and five for anti-lymphocyte antibodies. Anti-endothelial cell antibodies were common in patients in this group, being present in seven of eight individuals investigated.


Analgesic nephropathy and renal transplantation. Analgesic nephropathy was the most frequent diagnosis in 324 haemodialysis patients (30%) and the second most frequent diagnosis in 900 transplant patients (17%) at the Klinikum Steglitz, Berlin. Analgesic patients were the oldest group among haemodialysis and transplanted patients; the analgesic patients on haemodialysis were significantly older than the transplanted analgesic patients (55.9 +/- 11.2 versus 50.5 +/- 7.5 years, P = 10(-6)). Patient and graft survival under cyclosporin treatment are not statistically different in 125 transplantations of 109 patients with analgesic nephropathy compared to 508 transplantations of 423 patients with other renal diseases (5-year patient survival, 83.3 +/- 5.1% versus 88.4 +/- 2.4%; 5-year graft survival, 53.5 +/- 7.4% versus 56.8 +/- 3.8%; NS). Acetaminophen in urine was found in two of 30 analgesic and in one of 54 other patients with a functioning transplant (6.7% versus 1.9%; NS). As a sign of compliance, the mean cyclosporin trough level of 17 patients with analgesic nephropathy did not differ significantly from that of 14 patients with polycystic kidney disease (136.4 +/- 16.4 versus 139.4 +/- 17.3 ng/ml; NS), nor did the mean standard deviation of the individual measurements (36.7 +/- 36.8 versus 27.5 +/- 20.7 ng/ml; NS). Urothelial carcinoma was significantly more frequent in patients with analgesic nephropathy than in those with other renal diseases despite cystoscopy and retrograde pyelography before transplantation (3.7% versus 0.32%; P = 0.001). Vascular disease was the cause of death in 19 of 44 analgesic transplant patients who died (43.2%).(ABSTRACT TRUNCATED AT 250 WORDS)


Increased morbidity and mortality in patients with diabetes mellitus after kidney transplantation as compared with non-diabetic patients. The results of renal transplantation in patients with juvenile-onset diabetes mellitus were compared to those of a well-matched control group of non-diabetic patients. All transplantations were performed between 1977 and 1988. In the diabetic group hypertension (72 versus 41%), coronary artery disease (17 versus 0%), and peripheral vascular disease (19 versus 0%) had been significantly more frequent pretransplantation. Fewer diabetic patients had previously been treated with dialysis therapy (69 versus 97%). Graft function measured by creatinine clearance after 1 year follow-up, and incidence of proteinuria were not significantly different. The overall graft survival was significantly worse in the diabetic group compared to the control group: 42 versus 69% after 60 months and 21 versus 62% after 90 months. This was caused by a significantly worse patient survival in the diabetic group after 105 months: 28 versus 78% in the control group. The graft survival following exclusion of the patients who died with a functioning graft did not differ significantly between the groups after 60 and 90 months: 62 and 31% in the diabetic group and 69 and 62% in the control group. The existence of any vascular disease before transplantation, especially pre-existing peripheral vascular disease, had a significant effect on mortality in diabetic patients (P = 0.0003). After transplantation, diabetic patients had significantly more cerebrovascular accidents (23 versus 3%), peripheral vascular disease (31 versus 3%), and number of infections (1.9 versus 1.2). Retransplantation was carried out in each group to the same extent, with the same success rate.


Use of the urinary protein creatinine index to assess proteinuria in renal transplant patients. The use of 24-h urine protein collections for the assessment of proteinuria in renal transplant patients has been compared with the protein creatinine index (PCI) obtained from spot urine samples. Paired data from 148 patients showed a correlation of 0.77 (P less than 0.001) between 24-h protein excretion and the PCI. A PCI of 750 identified proteinuria of greater than 1.0 g/24 h with a sensitivity of 89% and a specificity of 93%. The predictive value of a positive test was 81% and that of a negative test 96%. Similar performance was observed for the detection of proteinuria of differing severities ranging from 0.5 g/24 h to 2.0 g/24 h. The use of spot testing was popular with both patients and staff, and reduced the sample handling cost to 15% of that of 24-h urine collection. We recommend that PCI be adopted as the standard outpatient test for the assessment of proteinuria following renal transplantation.


Attempts to make models for Alzheimer's disease. Profound reductions in cortical acetylcholine levels together with degeneration of cholinergic neurons in the basal forebrain have been reported in patients with Alzheimer's disease. A similar loss of the cholinergic neurons of the basal forebrain and impairment of learning and memory occur in animals injected with a nerve growth factor-diphtheria toxin conjugate, suggesting that this animal model is suitable to analyze cholinergic roles on learning and memory processes, and also the pathogenesis of Alzheimer's disease. In addition, animal models constructed by electrolytic or neurotoxic lesioning of the basal magnocellular nucleus, and models made by transgenetic technology were described.


Control of feeding rhythm in the terrestrial slug Incilaria bilineata. A modulatory neuron of feeding rhythm was newly identified in the buccal ganglia of the isolated central nervous system (CNS) of the terrestrial slug Incilaria bilineata. This neuron was termed the "feeding rhythm modulator" (FRM). Its morphological and electrical properties were compared with those of the MGC (metacerebral giant cell, a cerebral modulatory neuron of feeding rhythm). There was no direct connection between FRM and MGC. In order to investigate the control mechanism of the buccal central pattern generator, feeding rhythm was observed by varying the activities of MGC and FRM simultaneously. At a lower level of activity of MGC, feeding rhythm was not only sensitive to the activity of MGC but also to that of FRM. As the level of activity of MGC increased, feeding rhythm was exclusively controlled by the activity of MGC, and became unaffected by the activity of FRM. This indicates that cerebral neurons such as MGC primarily control feeding rhythm and modulate the contribution of FRM in a hierarchical manner.


Interleukin-6 as a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically defined medium from fetal and postnatal rat midbrains. Interleukin-6 (IL-6, human recombinant) promoted the survival of catecholaminergic neurons from fetal and postnatal rat midbrains as assessed by an immunohistochemical staining for tyrosine hydroxylase (TH) in culture using a chemically defined medium. The maximal dose of IL-6 for the cell survival of postnatal P15 rat mesencephalic TH-positive neurons in culture for 7 days was 50 ng/ml. The survival-promoting effects on P15 cultures were observed both in high- and low-density cultures. The survival effect of IL-6 on the cultured P15 TH-positive neurons was significant for only 4-15 days in vitro. However, the viable number of TH-positive neurons with IL-6 was less than that of the control at early points in the culture process (1-2 days in vitro). Continuous presentation of IL-6 was required for promoting survival. The optimal dose of IL-6 for the survival of fetal E16 midbrain TH-positive neurons was 5 ng/ml, and the survival promoting effect was less than that for the P15 cultures. The maximal dose of IL-6 for the survival of P2 TH-positive neurons was 5 ng/ml and that of P8 was 50 ng/ml, indicating that the response of rat mesencephalic TH-positive neurons to IL-6 changes during the first postnatal week.


Ocular torsion produced by unilateral chemical inactivation of the interstitial nucleus of Cajal in chronically labyrinthectomized cats. Unilateral lesions of the rostral midbrain involving the interstitial nucleus of Cajal (INC) have been known to produce ocular torsion in alert animals including humans, which has been assumed to be the result of an impaired otolith-ocular reflex. We examined the effects of chemical deactivation of the INC using a gamma-aminobutyric acid (GABA) agonist (muscimol) in cats that had received bilateral labyrinthectomy, and compared the results with those in normal cats. Ocular torsion with a magnitude similar to that observed in normal cats appeared in chronically labyrinthectomized cats after unilateral muscimol infusion into the INC, indicating that ocular torsion following unilateral INC deactivation can be produced by a mechanism independent of the otolith-ocular reflex.


GABA and behavior: the role of receptor subtypes. The discovery of different GABA receptor subtypes has stimulated research relating this neurotransmitter to a variety of behavioral functions and clinical disorders. The development of new and specific GABAergic compounds has made it possible to try to identify the specific functions of these receptors. The purpose of the present review is to evaluate the data regarding the functions of the GABA receptor subtypes in different behaviors such as motor function, reproduction, learning and memory, and aggressive-defensive behaviors. A description of GABAergic functions (stress, peripheral effects, thermoregulation) that might directly or indirectly affect behavior is also included. The possible involvement of GABA in different neurological and psychiatric disorders is also discussed. Although much research has been done trying to identify the possible role of GABA in different behaviors, the role of receptor subtypes has only recently attracted attention, and only preliminary data are available at present. It is therefore evident that still much work has to be done before a clear picture of the behavioral significance of these receptor subtypes can be obtained. Nevertheless, existing data are sufficient to justify the prediction that GABAergic agents, in the near future, will be much used in the field of behavioral pharmacology. It is hoped that the present review will contribute to this. Some specific suggestions concerning the most efficient way to pursue future research are also made.


Retinal pigment epithelium lesions as a biomarker of disease in patients with familial adenomatous polyposis. A follow-up report. The sensitivity of retinal pigment epithelium (RPE) lesions as a predictive congenital marker for the development of familial adenomatous polyposis (FAP) is evaluated. In a prospective study, 34 patients at 50% risk of inheriting FAP were examined. Based on the presence or absence of four or more RPE lesions, patients were categorized as those who had inherited or those who lacked the FAP genes. All patients received dilated fundus examinations with binocular indirect ophthalmoscopy and all RPE lesions were documented with fundus photography. All patients underwent annual sigmoidoscopy to determine the presence or absence of polyps. A 3-year follow-up analysis showed that 8 of 14 patients who were positive for RPE lesions later developed polyps. Of the 20 patients considered negative for FAP based on normal fundus examination, none has developed polyps. The authors urge all patient at risk of inheriting FAP to undergo dilated fundus examination with binocular indirect ophthalmoscopy and wide-angle fundus photography at the earliest age possible. All patients with 4 or more RPE lesions should undergo annual sigmoidoscopic examinations beginning before 10 years of age.


[Reactivity of hepatitis C virus (HCV) antibody among blood donors in Hungary]. The anti-HCV results of 1958 donors are discussed. The Elisa positivity of high risk subjects was as follows: 60% in coagulopathies, 14% in transfused haematological patients and 32% in patients on dialysis. Elisa reactivity of random blood donors was 1.82%, in the group, selected on the basis of transaminase positivity it was over 8%. The confirmed results were compared by the commercially available Elisa tests. 30 positive samples were confirmed by RIBA or Abbott confirmation assay. 35 of the 55 anti-HCV positives were reactive by all the Elisa, 19 samples could be confirmed, 25 were not subjected to any confirmatory assay. Further studies, and the introduction of mandatory screening of blood donors for anti-HCV are necessary.


[The role of HCV in the pathogenesis of post-transfusion hepatitis]. Between January 1987 and April 1988 following 462 open heart surgery operations, 83 patients with icteric hepatitis were seen. Among them 59 patients were anti-HCV positive with the Ortho anti-HCV test, these findings suggested a hepatitis C virus infection. The source of viral infection was searched, and a prothrombin complex concentrate which had been used during that period, seemed to be the potential cause of bloodborne hepatitis C. The results suggest that special care is required when using such blood products.


[Clinical and histological findings in HCV-caused hepatitis]. 58 cases of acute HCV-hepatitis were studied from the debut of acute illness and were followed up over 1-12 years. A mean three week's delay of anti-HCV IgG (Ortho) positivity was observed after the first manifestation of hepatitis. 70.6% relapsed and a chronic hepatitis developed in 37.9%. Seven patients died in HCV-hepatitis. Clinical, chemical, morphological features of HCV-hepatitis different from other viral hepatitis are demonstrated. Persistence of morphological signs of lobular hepatitis after even several months of subacute and chronic outcome has been found characteristic in HCV-hepatitis. Difficulties in interpretation of liver morphology are discussed. Simultaneous evaluation of clinical picture, laboratory markers and morphology is necessary to select cases for therapeutic trials.


[Transfusion-associated non-A, non-B, non-C hepatitis caused by flaviviruses]. Hepatitis C virus was shown to be a member of the flavivirus family. Tick-borne encephalitis virus and West Nile virus, members of the same family occur in Hungary, too. Serum samples from patients suffering from transfusion associated hepatitis were tested with yellow fever virus antigens for specific IgG, and IgM using immunofluorescence test. Eight hundred serum samples were tested. Yellow fever virus related IgG antibodies were found in 232 sera. In the case of 72 patients specific IgM antibodies could also be detected. The majority of the IgM positive patients underwent surgical operation and/or blood transfusion 1 to 2 months before the onset of the disease. Fifty-four sera positive for yellow fever virus-related antibodies were tested with HCV reagents, but only 13 were found to be positive, or cross-reacting. The 20 patients with yellow fever related antibodies were controlled with tick-borne encephalitis antigens, too. Nevertheless, no measurable cross-reaction could be detected. No measurable cross-reaction could be detected with the West Nile virus. The hepatitis B markers also were tested in 44 sera positive for yellow fever antibodies. There was only one, which contained HBsAg, and 10 of them proved to be positive for anti-HBcAg. The results indicate, that a non-A, non-B, non-C flavivirus is also present in the Hungarian population, which can be detected on the basis of the antigenic cross-reactivity with the attenuated yellow fever virus. This virus seems to be responsible for every 11th transfusion associated hepatitis examined.(ABSTRACT TRUNCATED AT 250 WORDS)


[Delta virus infection in hemodialyzed kidney patients and patients with chronic liver diseases]. Sera of patients with chronic hepatitis and chronic haemodialysed uraemia were tested for both Hepatitis B virus and Hepatitis Delta virus markers from 1985. Hepatitis Delta virus seroconversion was found in 10 of 88 haemodialysed patients and anti-Delta-IgG sero-positivity in 11 of 108 chronic hepatitis patients. The clinical course of Delta superinfections in haemodialysed patients was as follows: 3 infections were symptomless, 7 patients had acute hepatitis, two of the latter group died because of fulminant hepatitis. Of the 11 HBsAg and anti-Delta-IgG positive chronic hepatitis patients 3 had primary hepatocellular cancer.


[Hepatitis virus (HBV, HCV, HDV) markers in chronic liver diseases. Comparative studies in two East-Central European countries]. The prevalence of hepatitis virus markers in patients with chronic liver diseases from two countries has been studied: 68 patients (38 alcoholic hepatitis or cirrhosis, 30 chronic HBsAg-positive hepatitis) from Hungary as well as 109 patients (55 alcoholic liver disease, 45 chronic hepatitis or cryptogenic cirrhosis and 9 hepatoma) from Romania were examined for HBsAg, anti-HBs, anti-HBc, anti-HCV and anti-HDV, using the corresponding Abbott Elisa test systems. In alcoholic liver disease HBsAg occurred in 6/38 patients from Hungary and in 22/55 patients respectively, that is HBV markers occurred with significantly higher frequency in alcoholic patients from Romania (p less than 0.05). In the Hungarian group a total of 36 patients were HBsAg positive and out of them 5 had anti-HDV (13.9%), while out of 21 Romania HBsAg carriers 10 patients had anti-HDV (47.6%). Among 9 hepatoma patients 4 had HBsAg, 6 anti-HBs and 7 anti-HBc and 4 had anti-HCV and 3 had anti-HDV. One patient with hepatoma had both HBsAg and anti-HCV plus anti-HDV as well. Results suggest that the infection with hepatitis viruses in alcoholic liver diseases is more common in Romania than in Hungary, and the prevalence of delta virus infection in HBV carriers is also significantly higher in Romania than in Hungary.


[Acute viral hepatitis: pathogenesis and clinical manifestations]. This is a review of several mechanisms in the acute and chronic progress of viral hepatitis. Investigation of these mechanisms may have an important role in the diagnosis and treatment of chronic hepatitis. Clinical features of acute non-A, non-B hepatitis were studied in 110 patients. Most of the patients had a surgical intervention or/and received blood transfusion previously. The clinical course of the cases was very mild and most of the patients have the cholestatic form of the disease. A 39% anti-HCV antibody seropositivity rate was seen in these cases.


A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization. The p53 gene product has been implicated in both human and animal tumorigenesis. p53 forms heterologous complexes with the transforming proteins encoded by several different DNA tumor viruses. p53 also assembles into stable homo-oligomers. We demonstrate that the major structural determinant for the tetramerization of p53 is an alpha-helical plus basic region motif near the C-terminus of the protein. A monomeric p53 mutant adopts a conformation distinct from both 'wild-type' and 'mutant' form as defined by PAb1620 and PAb240 monoclonal antibody recognition. Nevertheless, monomeric and dimeric mutant p53 proteins retain the ability to suppress SV40 origin-directed DNA replication in vivo. Thus, p53-p53 interaction and expression of the PAb1620 epitope is not a prerequisite for such activity. We present data suggesting that suppression of replication by p53 may occur by a mechanism that is independent of detectable p53-T antigen association.


Functional interaction of p53 with HPV18 E6, c-myc and H-ras in 3T3 cells. Wild-type (wt) p53 has been suggested to be the product of a tumor-suppressor gene. Recently, it has been shown that the E6 oncoproteins of human papillomavirus (HPV) types 16 and 18, like the SV40 large T antigen, are physically associated with wt p53. We have investigated the functional interaction of wt p53 with the viral oncogene products of HPV16 and 18 and with cellular oncogenes by transfection of NIH3T3 cells with p53 wt alone or with several oncogene(s). We found that over-expression of HPV18 E6, c-myc or activated H-ras, like SV40 large T, can partially overcome the growth-inhibitory effect of wt p53 in NIH3T3 cells, while HPV16 E6 and E7, HPV18 E7, k-fgf, c-fos and mutant (mt) p53 do not. Further studies indicate that HPV18 E6 and c-myc can overcome the antiproliferative effect, but not the antitransforming effect, of wt p53, while activated H-ras can overcome both the antiproliferative and antitransforming effects of wt p53. These data show evidence of a functional interaction between HPV18 E6 and wt p53, and suggest that the cooperation of HPV E6 and cellular oncogenes c-myc and H-ras, which are activated in several cases of human cervical cancers, may be necessary to overcome completely the anti-oncogenic function of p53 in the development of these tumors.


Co-amplification of transcriptionally active epidermal growth factor receptor and ribosomal genes in the human hepatoma cell line Li7A. A high level of expression of the functional product of the epidermal growth factor receptor (EGFR) gene was detected in the human hepatocarcinoma cell line Li7A and it was found to correlate with gene amplification. The karyotype was paratriploid, with 15 rearranged chromosomes, several of which contained abnormally banded regions (ABRs). The search for DNA sequences co-amplified with the EGFR gene, using the in-gel renaturation technique, allowed us to detect an amplified DNA band (La1) of about 30 kb. This DNA was used as a probe for in situ hybridization on chromosomes, to locate the amplified segment. In normal lymphocytes, the DNA of band La1 hybridized to chromosome regions in which repetitive DNAs are located, i.e. on juxtacentromeric regions, the site of alphoid and CCATT satellite DNA, and on the short arms of acrocentrics, the site of ribosomal RNA (RNR) genes. In Li7A cells, it hybridized to the same regions and, in addition, to several chromosome arms corresponding to ABRs. The same ABRs hybridized to EGFR and RNR probes, but neither Alu sequences nor various probes for other repetitive sequences were recognized. They also exhibited nucleolus organizer region staining characterizing functionally active (RNR) genes. It was concluded that transcriptionally active genes were co-amplified in the same ABRs, although they originated from different chromosomes, i.e. chromosome 7 for EGFR and acrocentrics for RNR genes.


Mouse platelet-derived growth factor alpha receptor: sequence, tissue-specific expression and correlation with metastatic phenotype. High- and low-metastatic cells derived from metastatic murine tumors were screened for the differential expression of proto-oncogenes which may code for cell-surface receptors to growth factors. We found that metastatic clones of 3LL carcinoma and T10 sarcoma but not non-metastatic clones of these tumors express a 6.5-kb mRNA that is recognized by a v-fms probe containing a tyrosine kinase domain. The cloning and sequence analysis of a full-length cDNA clone corresponding to the v-fms-related 6.5-kb transcript showed that this transcript is the murine homolog of platelet-derived growth factor alpha (PDGF-alpha) receptor. The cDNA contains an open reading frame that predicts a 1089 amino acid protein. Comparison with the human and rat PDGF-alpha receptor reveals an overall amino acid sequence identity of 91% and 94% respectively. Northern blot analysis shows that this gene is preferentially expressed in the high-metastatic clones and is also selectively expressed in normal mouse tissues. Immunoprecipitation using anti-PDGF-alpha receptor serum shows that 185-kDa and 170-kDa proteins were specifically precipitated from cells of the high-metastatic D122 but not from the low-metastatic A9 cells. The possibility that overexpression of PDGF-alpha receptor in high-metastatic clones may contribute to an increase in the capacity of tumor cells to generate metastases in the lung is discussed.


Slow time-dependent cellular transformation induced at restrictive temperature by ts-src mutants. Cell lines infected with a temperature-sensitive Rous sarcoma virus have been widely used to study the temporal dynamics of various transformation parameters following downshift from the non-permissive temperature to the permissive temperature as it is considered that, at the non-permissive temperature, the infected cells exhibit the morphological and growth characteristics of normal cell whereas, at the permissive temperature, they exhibit characteristics of the transformed state. We show here that the apparently normal state in which tsPA1-infected FR3T3 cells are directed at the restrictive temperature is not a stationary stable state, but rather a transient one which continuously drifts as the cells are grown and passaged at this temperature and which eventually ends up as a new transformed state (T2) with morphological and growth properties definitely different from those belonging to the transformed state (T1) at the permissive temperature. The establishment of the transformed T2 state at the restrictive temperature occurs concomitantly with a steady accumulation of an intracellular pool of pp60v-src in the vicinity of the nucleus, whose traffic towards the plasma membrane is released following downshift to the permissive temperature, leading to the reappearance of transformation parameters characteristic of the transformed T1 state. Our finding raises the possibility that the v-src protein encoded by the tsPA1 mutant of Rous sarcoma virus may induce cellular transformation via two different pathways, leading to two different transformation states, depending on at which temperature the infected cells are grown. Various possible mechanisms that could be involved in the time-dependent establishment of a transformed state by ts-src mutants at the restrictive temperature are discussed.


Modification of cyclin A expression by hepatitis B virus DNA integration in a hepatocellular carcinoma. We have previously reported the identification of a hepatitis B virus (HBV) DNA integration in an intron of the cyclin A gene in an early hepatocellular carcinoma (HCC) and the isolation of human cyclin A cDNA. We have now constructed a cDNA library from the tumor and isolated several hybrid HBV-cyclin A cDNAs from it. The hybrid cDNAs encode an HBV-cyclin A fusion protein. In the chimeric protein, the N-terminus of cyclin A, including the signals for cyclin degradation, is deleted and replaced by viral PreS2/S sequences, transcription being initiated from the viral PreS2/S promoter. This chimeric protein is undegradable in an in vitro cyclin degradation assay. Northern blot analyses showed strong expression of the hybrid transcripts in the tumor, while cyclin A- or HBV-specific transcripts were not detected in the non-tumorous liver of the same patient. Thus, HBV DNA integration in the cyclin A gene resulted in a strong expression of hybrid HBV-cyclin A transcripts encoding a stabilized cyclin A. This chimeric protein may play an important role in the development of the tumor.


TTX-sensitive Na+ channels and Ca2+ channels of the L- and N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats. Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na+ currents revealed that tetrodotoxin (TTX, 1 microM) completely inhibited the large transient inward current. Half activation potential (Vh) and slope factor (K) were -26.8 mV and 6.1 mV, respectively. Inactivation parameters for Vh and K were -65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca2+ current which was blocked by the removal of external Ca2+ or the presence of Cd2+ (0.1 mM). The dihydropyridine agonist, (+)202-791 (1 microM), caused a small increase (20%) in the amplitude of the Ca2+ current at more negative potentials and markedly prolonged the tail currents. omega-Conotoxin GIVA (omega, CgTX, 15 microM) caused a 66% inhibition of the high-voltage-activated Ca2+ current amplitude. Norepinephrine (1 microM) caused a 49% reduction in the peak Ca2+ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na+ current as well as a high voltage-activated sustained Ca2+ current underlie the inward current in C-SMG neurons. The macroscopic Ca2+ current is composed of a small dihydropyridine-sensitive (L-type current) and a large omega-CgTx-sensitive (N-type current) component.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells. Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4 alpha-phorbol ester 4 alpha-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The beta-adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[gamma S], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the beta 1 receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the beta receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells.


[MR imaging of hilar cholangiocarcinoma--comparative study with CT]. Magnetic resonance (MR) images of 18 cases of hilar cholangiocarcinoma were evaluated to compare the effectiveness of Gd-DTPA with that of high dose contrast enhancement computed tomography (HCE-CT) in detecting the primary tumor. The primary tumor was demonstrated as having slightly low intensity compared with liver parenchyma and high intensity compared with the dilated bile duct on T1 weighted images. In contrast, MRI using Gd-DTPA, which was carried out in five cases, revealed intense enhancement of the tumor. As the differentiation between cholangiocarcinoma and dilated bile duct was difficult, it was concluded that the use of Gd-DTPA improves the efficacy of MRI in diagnosing cholangiocarcinoma. Gd-DTPA was also effective in differentiating the growth pattern of the tumor: the infiltrating type was demonstrated as thickening of the wall of the bile duct, the polypoid type as a soft tissue mass in the bile duct. Contrast MRI study is effective for the detection of cholangiocarcinoma. It is also expected to be effective in the staging diagnosis of cholangiocarcinoma.


The AT-rich tract of the SV40 ori core: negative synergism and specific recognition by single stranded and duplex DNA binding proteins. The SV40 origin of replication comprises a run of thymine and adenine residues. Integrity of this AT-rich sequence is known to be essential for replication. We set out to study whether or not these elements can work synergistically to sustain replication. Quite surprisingly, additional copies of the AT stretch linked to a functional SV40 ori core dramatically reduce its replication in Cosl cells, probably by creating some physical block. Interestingly, the same inhibiting effect can be observed with the addition in cis of the yeast ARS consensus, which is homologous to the SV40 AT stretch. This modulation is possibly due to the action of cellular factors that recognize either of the two sequences. In fact, we demonstrate the existence of factor(s) in Cosl crude nuclear extracts that in vitro can specifically bind to either of them. Moreover, we show that these sequence-specific factor(s) (MW about 50 kDa), named SOAP, recognize both single (T-rich strand) and double stranded forms of the AT tracts. Binding to single stranded AT stretches can be specifically inhibited by the corresponding duplex form, but not vice versa.


Topoisomerase I is preferentially associated with normal SV40 replicative intermediates, but is associated with both replicating and nonreplicating SV40 DNAs which are deficient in histones. Based on the use of equilibrium centrifugation in CsCl to separate covalent complexes between topoisomerase I and DNA from protein-free DNA, it was concluded previously that the topoisomerase is preferentially associated with replicating SV40 DNA (Champoux, J. J. 1988. J. Virol. 62:3675-3683). One explanation for the failure to find the enzyme associated with nonreplicating viral DNA is that most of the completed DNA is rapidly sequestered for encapsidation and inaccessible to topoisomerase I. This explanation has been ruled out in the present work by the finding that topoisomerase I in COS-1 cells is also preferentially associated with the replicative form of an SV40 origin-containing plasmid that lacks the genes coding for the virion structural proteins and therefore cannot be encapsidated. Thus it appears that some structural feature of the replicating DNA or the replication complex specifically recruits the topoisomerase to the DNA. SV40 DNA which is produced in the presence of the protein synthesis inhibitor, puromycin, is deficient in histones and as a result lacks normal chromatin structure. Topoisomerase I was found to be associated with SV40 DNA under these conditions whether or not it was replicating. This observation is interpreted as an indication that under normal conditions, chromatin structure limits access of topoisomerase I to the nonreplicating viral DNA.


Kinetoplast-associated DNA topoisomerase in Crithidia fasciculata: crosslinking of mitochondrial topoisomerase II to both minicircles and maxicircles in cells treated with the topoisomerase inhibitor VP16. The mitochondrial DNA of the trypanosomatid Crithidia fasciculata consists of thousands of copies of a 2.5 kb minicircle and a small number of 37kb maxicircles catenated into a single enormous network. Treatment of C. fasciculata with the type II DNA topoisomerase inhibitor VP16 produces cleavable complexes of a type II DNA topiosomerase with both minicircles and maxicircles. A combined Southern and Western blot analysis of the cleaved DNA species released from the network by SDS treatment has identified topollmt, the kinetoplast-associated topisomerase, in covalent complexes with linear forms of minicircle and maxicircle DNAs. These results directly implicate topollmt in the topological reactions required for the duplication of the kinetoplast network.


Mammalian promoter element function in the fission yeast Schizosaccharomyces pombe. We have analyzed the function of several mammalian promoter elements in the fission yeast, Schizosaccharomyces pombe. Mutants of the human HSP70 promoter were introduced into S. pombe as single copy integrants at a specific location. Transcription initiation sites utilized in S. pombe with the HSP70 TATA element were similar to those used in mammalian cells. Of three mammalian TATA elements tested, only the HSP70 TATA element functioned in S. pombe. The adenovirus Ella TATA element had little or no activity in S. pombe, indicating that S. pombe is deficient in the factor(s) necessary for recognition of this element. Of upstream promoter elements tested, the CCAAT, Sp1 binding, ATF binding and heat shock elements were functional in S. pombe. Strains containing mutant promoters fused to the ble gene were used to demonstrate that phleomycin can be used as a graded selection in S. pombe. These data demonstrate that S. pombe should provide a useful system in which to characterize and isolate mammalian factors involved in initiation site determination and transcriptional regulation.


The prediction of exons through an analysis of spliceable open reading frames. We have developed a computer program which predicts internal exons from naive genomic sequence data and which will run on any IBM-compatible 80286 (or higher) computer. The algorithm searches a sequence for 'spliceable open reading frames' (SORFs), which are open reading frames bracketed by suitable splice-recognition sequences, and then analyzes the region for codon usage. Potential exons are stratified according to the reliability of their prediction, from confidence levels 1 to 5. The program is designed to predict internal exons of length greater than 60 nucleotides. In an analysis of 116 genes of a training set, 384 out of 441 such exons (87.1%) are identified, with 280 (63.5%) of predictions matching the true exon exactly (at both 5' and 3' splice junctions and in the correct reading frame), and with 104 (23.6%) exons matching partially. In a similar analysis of 14 genes in a test set unrelated to the genes used to generate the parameters of the program, 70 out of 80 internal exons greater than 60 bp in length are identified (87.5%), with 47 completely and 23 partially matched. SORFs that partially match true internal exons share at least one splice junction with the exon, or share both splice junctions but are interpreted in an incorrect reading frame. Specificity (the percentage of SORFs that correspond to true exons) varies from 91% at confidence level 1 to 16% at confidence level 5, with an overall specificity of 35-40%. The output displays nucleotide position, confidence level, reading frame phase at the 5' and 3' ends, acceptor and donor sequences and scoring statistics and also gives an amino acid translation of the potential exon. SORFIND compares favourably with other programs currently used to predict protein-coding regions.