Insulin receptor gene expression during development: developmental regulation of insulin receptor mRNA abundance in embryonic rat liver and yolk sac, developmental regulation of insulin receptor gene splicing, and comparison to abundance of insulin-like growth factor 1 receptor mRNA. Insulin gene expression has been demonstrated in nonpancreatic tissues early in development, suggesting that this hormone might have actions significant for the differentiating embryo. Because such actions imply ligand-receptor binding, we quantified mRNAs encoding the two known forms of insulin receptor in rat liver and yolk sac, two endodermally derived tissues shown to express insulin genes, between gestation days (E) 13 and E21 (mid-organogenesis to parturition). Because of its presumed importance for fetal growth, we estimated the abundance of mRNA encoding insulin-like growth factor 1 (IGF 1) receptor in the same samples for comparison. The abundance of insulin receptor mRNA exceeded that for IGF 1 receptor mRNA in liver and yolk sac at all times studied. This difference was greater in liver, where insulin receptor mRNAs were three to more than 50 times more abundant than IGF 1 receptor mRNA on gestation days E13-E16, times which antedate the development of significant hepatic metabolic actions of insulin. The marked abundance of mRNAs encoding insulin receptors is consistent with the hypothesis that insulin has significant actions in specific tissues during the organogenic period.


Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity. The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.


Characterization of the genomic corticotropin-releasing factor (CRF) gene from Xenopus laevis: two members of the CRF family exist in amphibians. In mammals, the release of pituitary ACTH is stimulated by CRF. Two related peptides exist in nonmammalian vertebrates, sauvagine from frog skin and urotensin-I from the urophysis of teleost fish. Their related structures (approximately 50%) and capacity to stimulate the release of ACTH from mammalian and fish pituitaries has led to the proposal that sauvagine and urotensin-I are homologs of mammalian CRF. However, sauvagine does not appear to stimulate ACTH release in amphibians, although mammalian CRF (ovine) induces a potent response from amphibian pituitaries. This could indicate that the main function of sauvagine does not involve ACTH regulation and suggests that an additional CRF-like peptide exists in Amphibia. We report here the isolation of two highly homologous CRF-like genes from the frog, Xenopus laevis. Analysis of the expression pattern of these CRF-like genes revealed mRNA in splenic tissue and in the preoptic nucleus and paraventricular organ of the brain. The amino acid sequence of the mature peptide regions (1-41) of both X. laevis genes is strikingly conserved, sharing more than 93% homology with mammalian CRFs, yet only 50% homology with sauvagine. In view of the fact that these new amphibian CRF-like genes share far greater homology with mammalian CRF than that exhibited by sauvagine, we propose that the new Xenopus CRF-like genes are the amphibian counterparts to mammalian CRF. Thus, two members of the CRF family have now been identified in the Amphibia, namely CRF and sauvagine.


Promoter for the regulatory type I beta subunit of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase directs transgene expression in the central nervous system. Cyclic AMP-dependent protein kinase (cAPK) modulates synaptic transmission and influences memory and learning. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian cAPK, only the regulatory type I beta (RI beta) subunit is unique to nervous tissue. The requirement for RI beta in neurons is presently unknown. Previous studies demonstrate that holoenzyme containing RI beta activates at lower concentrations of cAMP compared to other forms of cAPK. Thus, neurons that induce RI beta expression may become more sensitive to subsequent hormonal signals and maintain more long-term phosphorylation events. To further elucidate the function of this novel protein, we have begun to investigate its gene. Here we report the isolation of the mouse RI beta promoter as determined by S1 nuclease analysis and transgenic mouse expression. A beta-galactosidase fusion gene containing 1.5 kilobases of 5'-nontranscribed RI beta DNA and 2 kilobases of intron 1 was expressed preferentially in the cortex and hippocampus of the brain and within the spinal cord. In addition to mimicking the location of endogenous RI beta expression, the transgene was activated at a similar time (embryonic day 11.5) during mouse fetal development. Isolation of the RI beta promoter will help identify the elements that direct transcription in a subset of neurons and illuminate the physiological conditions that may regulate RI beta expression. This promoter can also be used to target the expression of wild type and mutant cAPK subunit genes in order to investigate synaptic plasticity in animals.


An association between collateral blood flow and myocardial viability in patients with recent myocardial infarction. We hypothesized that successful reperfusion of an occluded infarct-related coronary artery even late after acute myocardial infarction would result in improved regional wall motion and that such improvement might be related to the presence of collateral blood flow within the infarct bed. We assessed regional wall motion by two-dimensional echocardiography at base line and one month after angioplasty was attempted in the occluded infarct-related artery in 43 patients who had had a myocardial infarction two days to five weeks earlier. A wall-motion score was assigned to each patient on a five-point scale (from 1 [normal function] to 5 [dyskinesia]). The percentage of the infarct bed perfused by collateral flow was assessed with myocardial contrast echocardiography. In the 41 patients who had abnormal wall motion at base line, improvement in function was noted in 25 (78 percent) of the 32 in whom angioplasty was successful, as compared with only 1 (11 percent) of the 9 in whom it was unsuccessful (P < 0.001). The percentage of the infarct bed supplied by collateral flow at base line was directly correlated with wall function and inversely correlated with the wall-motion score one month after successful angioplasty (r = -0.64, P < 0.001). Among the patients in whom angioplasty was successful, the 23 in whom > 50 percent of the infarct bed was supplied by collateral flow had better wall motion (P < 0.001) and greater improvement in wall motion at one month (P = 0.004) than the 9 in whom < or = 50 percent of the bed was supplied by collateral flow. The degree of improvement in function was not influenced by the length of time between the infarction and the attempted angioplasty. The myocardium remains viable for a prolonged period in many patients with acute infarction and an occluded infarct-related artery. Viability appears to be associated with the presence of collateral blood flow within the infarct bed.


A controlled trial of trimethoprim-sulfamethoxazole or aerosolized pentamidine for secondary prophylaxis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. AIDS Clinical Trials Group Protocol 021. Pneumocystis carinii pneumonia (PCP) continues to be the most common index diagnosis in the acquired immunodeficiency syndrome (AIDS), but it is not clear which of several available agents is the most effective in preventing a recurrence of PCP. We conducted a comparative, open-label trial in 310 adults with AIDS who had recently recovered from an initial episode of PCP and had no treatment-limiting toxic effects of trimethoprim-sulfamethoxazole or pentamidine. All the patients were treated with zidovudine and were randomly assigned to receive either 800 mg of sulfamethoxazole and 160 mg of trimethoprim once daily or 300 mg of aerosolized pentamidine administered every four weeks by jet nebulizer. The participants were followed for a median of 17.4 months. In the trimethoprim-sulfamethoxazole group (n = 154) there were 14 recurrences of PCP, as compared with 36 recurrences (including 1 extrapulmonary recurrence) in the aerosolized-pentamidine group (n = 156). The estimated recurrence rates at 18 months were 11.4 percent with trimethoprim-sulfamethoxazole and 27.6 percent with pentamidine (P < 0.001). The risk of a recurrence (adjusted for initial CD4 cell count) was 3.25 times higher in the pentamidine group (P < 0.001, 95 percent confidence interval, 1.72 to 6.16). There were no significant differences between the groups in survival or in hematologic or hepatic toxicity. Crossovers from trimethoprim-sulfamethoxazole to aerosolized pentamidine were more common than the reverse (27 vs. 4 percent), partly because of the study protocols for the management of leukopenia. There were 19 serious bacterial infections in the trimethoprim-sulfamethoxazole group and 38 in the pentamidine group. The time to a first bacterial infection was significantly greater for those assigned to trimethoprim-sulfamethoxazole (P = 0.017). In patients with AIDS who are receiving zidovudine, trimethoprim-sulfamethoxazole is more effective than aerosolized pentamidine in conventional doses for the prevention of recurrent pneumocystis infection.


The self-limited nature of chronic idiopathic diarrhea. Little is known about the clinical presentation and natural history of previously healthy patients in whom chronic idiopathic diarrhea develops. We reviewed the case records of 152 patients with chronic diarrhea who had no history of gastrointestinal surgery and who were evaluated in detail as part of a chronic-diarrhea protocol from 1985 to 1990. Patients were considered to have chronic idiopathic diarrhea if they had persistently loose stools for more than four weeks, no systemic illness, and no identifiable cause of diarrhea. Seventeen patients (10 men and 7 women) ranging in age from 33 to 72 years met the criteria for chronic idiopathic diarrhea. Each patient had a history of a relatively abrupt onset of symptoms, often soon after returning home from a trip, starting two to seven months before evaluation. Their diarrhea did not occur during a local outbreak of diarrhea, and other family members did not become ill. Stool frequency ranged from 5 to 25 movements per day, stool weights ranged from 417 to 1480 g per day, and fecal electrolyte and osmolality values were consistent with a diagnosis of secretory diarrhea. The results of biopsies of the small intestine and colon were normal, as were small-bowel roentgenograms. Extensive studies for infectious causes of diarrhea were negative, and no patient responded to antibiotic therapy. In every patient the diarrhea stopped without specific therapy after 7 to 31 months (mean, 15) and did not recur during a follow-up period averaging 38 months. Sporadic idiopathic chronic diarrhea is a recognizable syndrome that can last many months, but is self-limited.


Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. The inhibins are alpha:beta heterodimeric growth factors that are members of the transforming growth factor-beta family. To understand the physiological roles of the inhibins in mammalian development and reproduction, a targeted deletion of the alpha-inhibin gene was generated by homologous recombination in mouse embryonic stem cells. Mice homozygous for the null allele (inhibin-deficient) initially develop normally but every mouse ultimately develops mixed or incompletely differentiated gonadal stromal tumours either unilaterally or bilaterally. Inhibin is thus a critical negative regulator of gonadal stromal cell proliferation and the first secreted protein identified to have tumour-suppressor activity.


Functional localization and lateralization of human olfactory cortex. Anatomical and physiological investigations in monkeys indicate that olfaction is subserved by several cortical regions. But the areas implicated in the human olfactory system have not been definitively identified by functional criteria. Behavioural evidence has suggested that laterally specialized mechanisms for odour processing may exist, but the neuroanatomical substrate remains unknown. We used positron emission tomography to study the cortical representation of human olfactory processing by comparing cerebral blood flow changes evoked during olfactory stimulation with those of a control task. We report here significant cerebral blood flow increases at the junction of the inferior frontal and temporal lobes bilaterally, corresponding to the piriform cortex, and unilaterally, in the right orbitofrontal cortex. The results complement and extend previous data implicating these regions in olfactory processing, and indicate that a functional asymmetry exists in the human brain favouring the right orbitofrontal area in olfaction.


Columns for visual features of objects in monkey inferotemporal cortex. At early stages of the mammalian visual cortex, neurons with similar stimulus selectivities are vertically arrayed through the thickness of the cortical sheet and clustered in patches or bands across the surface. This organization, referred to as a 'column', has been found with respect to one-dimensional stimulus parameters such as orientation of stimulus contours, eye dominance of visual inputs, and direction of stimulus motion. It is unclear, however, whether information with extremely high dimensions, such as visual shape, is organized in a similar columnar fashion or in a different manner in the brain. Here we report that the anterior inferotemporal area of the monkey cortex, the final station of the visual cortical stream crucial for object recognition, consists of columns, each containing cells responsive to similar visual features of objects.


Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. The fungal metabolite brefeldin A is a powerful tool for investigating membrane traffic in eukaryotic cells. The effects of brefeldin A on traffic are partly explained by its ability to prevent binding of cytosolic coat proteins onto membranes. The non-clathrin coatomer complex binds reversibly to Golgi membranes in a GTP-controlled cycle. The low-molecular-mass GTP-binding protein ADP-ribosylation factor (ARF), which also associates reversibly with Golgi membranes, is required for coatomer binding and probably accounts for the control by guanine nucleotide of the coatomer-membrane interaction. Brefeldin A prevents the assembly of coatomer onto the membrane by inhibiting the GTP-dependent interaction of ARF with the Golgi membrane, but the nature of this interaction has not been established. Here we demonstrate that Golgi membranes can specifically catalyse the exchange of GTP onto ARF and that brefeldin A prevents this function.


Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4), implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles, beta-COP, implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains, one of which is beta-COP), the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein, the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al., manuscript submitted), so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed, like beta-COP, ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro, but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein, and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways.


Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle. We report here the determination and refinement to 1.9 A resolution by X-ray cryo-crystallography the structure of HLA-Aw68. The averaged image from the collection of bound, endogenous peptides clearly shows the atomic structure at the first three and last two amino acids in the peptides but no connected electron density in between. This suggests that bound peptides, held at both ends, take alternative pathways and could be of different lengths by bulging out in the middle. Peptides eluted from HLA-Aw68 include peptides of 9, 10 and 11 amino acids, a direct indication of the length heterogeneity of tightly bound peptides. Peptide sequencing shows relatively conserved 'anchor' residues at position 2 and the carboxy-terminal residue. Conserved binding sites for the peptide N and C termini at the ends of the class I major histocompatibility complex binding groove are apparently dominant in producing the long half-lives of peptide binding and the peptide-dependent stabilization of the class I molecule's structure.


Atomic structure of a human MHC molecule presenting an influenza virus peptide. Infection by influenza virus results in the stimulation of cytotoxic T lymphocytes specific for killing virally infected cells. Specificity is provided by clonally distributed, hypervariable T-cell receptors on cytotoxic T lymphocytes which react with peptide fragments that are derived from viral proteins expressed in the cytoplasm and 'presented' on the surface of infected cells, bound to class I histocompatibility glycoproteins. Here we describe the structure of the complex between the human class I histocompatibility glycoprotein HLA-Aw68 and the influenza virus nucleoprotein peptide Np 91-99 as determined by X-ray cryocrystallography. Residues at both ends of the peptide are substantially buried in the peptide binding-site, whereas those in the middle of the peptide, P4 to P8, are predominantly exposed and could be recognized directly by T-cell receptors. The extended conformation of the bound viral peptide is remarkably similar to that of a collection of endogenous peptides with a different sequence motif bound to another human allele, HLA-B27. The structure defines in atomic detail the antigenic surface constructed of major histocompatibility complex and viral peptide atoms that is recognized by T-cell receptors.


The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.


AIDS and sexual behaviour in France. ACSF investigators. The results of a massive telephone survey of sexual lifestyles in France should provide a basis for prevention strategies for AIDS and sexually transmitted diseases.


Sexual lifestyles and HIV risk.

Sexual lifestyles and HIV risk. Britain's first large, national survey of sexual attitudes and lifestyles will allow improved estimates of the magnitude of the HIV epidemic in Britain and should lead to better strategies for prevention.


Production of sperm reduces nematode lifespan. Sex and death are two fundamental but poorly understood aspects of life. They are often thought to be linked because reproduction requires the diversion of limited resources from somatic growth and maintenance. This diversion of resources in mated animals, often called a cost of reproduction, is usually expressed as a reduction of lifespan in mated animals, although some debate exists on the best way to measure this cost. I report here that in the soil nematode, Caenorhabditis elegans, sex significantly decreases male lifespan without reducing hermaphrodite lifespan. The reduction of mated male lifespan seems to be caused by additional sperm production and not by the physical activity of mating. This conclusion is supported by observations that a mutation reducing sperm production increased mean lifespan by about 65% in both mated males and hermaphrodites. This suggests that spermatogenesis, rather than oogenesis or the physical act of mating, is a major factor reducing lifespan in C. elegans. This contradicts the traditional biological assumption that large oocytes are much costlier to produce than small sperm.


Viscous fingering of HCl through gastric mucin. The HCl in the mammalian stomach is concentrated enough to digest the stomach itself, yet the gastric epithelium remains undamaged. One protective factor is gastric mucus, which forms a protective layer over the surface epithelium and acts as a diffusion barrier Bicarbonate ions secreted by the gastric epithelium are trapped in the mucus gel, establishing a gradient from pH 1-2 at the lumen to pH 6-7 at the cell surface. How does HCl, secreted at the base of gastric glands by parietal cells, traverse the mucus layer without acidifying it? Here we demonstrate that injection of HCl through solutions of pig gastric mucin produces viscous fingering patterns dependent on pH, mucin concentration and acid flow rate. Above pH 4, discrete fingers are observed, whereas below pH 4, HCl neither penetrates the mucin solution nor forms fingers. Our in vitro results suggest that HCl secreted by the gastric gland can penetrate the mucus gel layer (pH 5-7) through narrow fingers, whereas HCl in the lumen (pH 2) is prevented from diffusing back to the epithelium by the high viscosity of gastric mucus gel on the luminal side.


Possible blindsight in infants lacking one cerebral hemisphere. Patients with damage to the striate cortex have a subjectively blind region of the visual field, but may still be able to detect and localize targets within this region. But the relative roles in this 'blindsight' of subcortical neural systems, and of pathways to extra-striate visual areas, have been uncertain. Here we report results on two infants in whom one cerebral hemisphere, including both striate and extra-striate visual cortex, needed surgical removal in their first year. Single conspicuous targets in the half-field contralateral to the lesion could elicit fixations, implying detection and orienting by a subcortical system. In contrast, binocular optokinetic nystagmus (OKN), for which a subcortical pathway has often been thought adequate, showed a marked asymmetry. In normal neonates, fixation shifts and OKN have both been taken to reflect subcortical control; our results are consistent with subcortical control for fixation but not for OKN.


Cloning and expression of a rat brain L-glutamate transporter. Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.


The glial cell glutamate uptake carrier countertransports pH-changing anions. Uptake into glial cells helps to terminate glutamate's neurotransmitter action and to keep its extracellular concentration, [Glu]o, below neurotoxic levels. The accumulative power of the uptake carrier stems from its transport of inorganic ions such as sodium (into the cell) and potassium (out of the cell). There is controversy over whether the carrier also transports a proton (or pH-changing anion). Here we show that the carrier generates an alkalinization outside and an acidification inside glial cells, and transports anions out of the cells, suggesting that there is a carrier cycle in which two Na+ accompany each glutamate anion into the cell, while one K+ and one OH- (or HCO3-) are transported out. This stoichiometry predicts a minimum [Glu]o of 0.6 microM normally (tonically activating presynaptic autoreceptors and post-synaptic NMDA receptors), and 370 microM during brain anoxia (high enough to kill neurons). Transport of OH-/HCO3- on the uptake carrier generates significant pH changes, and may provide a mechanism for neuron-glial interaction.


HLA-DR molecules from an antigen-processing mutant cell line are associated with invariant chain peptides. The invariant chain, which associates with the major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, serves two functions important in antigen processing. First, it prevents class II molecules from binding peptides in the early stages of intracellular transport. Second, it contains a cytoplasmic signal that targets the class II-invariant chain complex to an acidic endosomal compartment. Proteolytic cleavage and subsequent dissociation of the invariant chain then occurs, allowing peptides derived from endocytosed proteins to bind to released class II molecules before their expression at the cell surface. Certain human cell lines that are mutant in one or more MHC-linked genes are defective in class II-restricted antigen processing. Here we show that in transfectants of one of these cell lines, T2, this deficiency results in the association of a large proportion of class II molecules with a nested set of invariant-chain-derived peptides (class II-associated invariant chain peptides, or CLIP). HLA-DR3 molecules isolated from T2 transfectants can be efficiently loaded with antigenic peptides by exposure to a low pH in vitro, perhaps reflecting the in vivo conditions in which peptides associate with class II molecules. Addition of synthetic CLIP inhibits the loading process, indicating that CLIP may define the region of the invariant chain responsible for obstructing the class II binding site.


Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes. The co-ordinated function of effector and accessory cells in the immune system is assisted by adhesion molecules on the cell surface that stabilize interactions between different cell types. Leukocyte function-associated antigen 1 (LFA-1) is expressed on the surface of all white blood cells and is a receptor for intercellular adhesion molecules (ICAM) 1 and 2 (ref. 3) which are members of the immunoglobulin superfamily. The interaction of LFA-1 with ICAMs 1 and 2 provides essential accessory adhesion signals in many immune interactions, including those between T and B lymphocytes and cytotoxic T cells and their targets. In addition, both ICAMs are expressed at low levels on resting vascular endothelium; ICAM-1 is strongly upregulated by cytokine stimulation and plays a key role in the arrest of leukocytes in blood vessels at sites of inflammation and injury. Recent work has indicated that resting leukocytes express a third ligand, ICAM-3, for LFA-1 (refs 11, 12). ICAM-3 is potentially the most important ligand for LFA-1 in the initiation of the immune response because the expression of ICAM-1 on resting leukocytes is low. We report the expression cloning of a complementary DNA, pICAM-3, encoding a protein constitutively expressed on all leukocytes, which binds LFA-1. ICAM-3 is closely related to ICAM-1, consists of five immunoglobulin domains, and binds LFA-1 through its two N-terminal domains.


Cloning and characterization of a new intercellular adhesion molecule ICAM-R. The human intercellular adhesion molecules ICAM-1, ICAM-2 and their counter-receptors, the beta 2 or leukointegrins, mediate a variety of homotypic and heterotypic leukocyte and endothelial cell-cell adhesions central to immunocompetence. It has been found that cell-cell adhesion which is dependent on expression of the leukocyte function-associated antigen LFA-1 is not always blocked completely by antibodies raised against ICAM-1 and ICAM-2. Other leukointegrin ligands therefore probably exist, such as a glycoprotein of M(r) 124K that binds LFA-1 and has been designated ICAM-3 on the basis of this function. We have molecularly cloned a new member of the ICAM family, ICAM-R, which is related to ICAM-1 and ICAM-2. The complementary DNA encoding ICAM-R is 1,781 base pairs long and the protein has five extracellular immunoglobulin-family type domains. The mature cell-surface form of the ICAM-R protein has an M(r) which varies from 116 to 140K in a cell type-specific fashion. Overall identities in protein sequence with ICAM-1 and ICAM-2 are 48% and 31% respectively, with the degree of similarity varying between individual domains. The high level of expression of ICAM-R on resting leukocytes of all lineages and its lack of expression on either resting or cytokine-activated endothelial cells indicates a pattern of expression distinct from ICAM-1 and ICAM-2. In common with ICAM-1 and ICAM-2, ICAM-R is a ligand for the beta 2-integrin CD11a/LFA-1 (CD18).


Imprinting and splicing join together.

Imprinting and splicing join together. A gene thought to be involved in RNA splicing, and which is imprinted in mice, may play an important part in Prader-Willi syndrome.


Making light work with optical tweezers. Microscopic objects, including biological material, can be remotely manipulated with tightly focused beams of infrared laser light. The use of optical traps, or 'optical tweezers', holds great promise for noninvasive micromanipulation and mechanical measurement in cell biology. Optical tweezers are the 'tractor beams' of today's technology.


Long lasting attenuation of 8-OH-DPAT-induced corticosterone secretion after a single injection of a 5-HT1A receptor agonist. The effects of 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) and some other 5-hydroxytryptamine1A (5-HT1A) receptor agonists (buspirone, ipsapirone and flesinoxan) on corticosterone secretion in rats were studied. The 5-HT1A receptors mediating the corticosterone secretion appear to be postsynaptic to the 5-HT neurons, since the response to 8-OH-DPAT was not decreased but potentiated by depletion of 5-HT with p-chlorophenylalanine pretreatment of the animals. Rapid attenuation of the response was developed after a single dose of a 5-HT1A receptor agonist. Thus, 1 mg/kg s.c. of 8-OH-DPAT attenuated the response of a challenge dose (0.1 mg/kg s.c.) of this compound within 4 h lasting between 7 and 14 d. The development of the subsensitivity was antagonized by pretreatment of the rats with the 5-HT1A receptor antagonist S-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((-)-UH 301). This compound also antagonized the acute effect of 8-OH-DPAT in increasing serum corticosterone. The subsensitivity development was specific for the 5-HT1A receptor-mediated corticosterone secretion, since the increase in serum corticosterone produced by stimulation of other receptor systems, e.g. alpha 2-adrenoreceptors (clonidine) or 5-HT2 receptors [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, (DOI)] was not affected.


Functional expression of human 5-HT1A receptors and differential coupling to second messengers in CHO cells. The signal transduction linkages of the cloned human 5-HT1A receptor as expressed stably in CHO cells were studied. A transfected clonal cell line which expresses 900 +/- 36 fmol 5-HT1A receptor/mg protein (designated CHO-5-HT1A/WT-27) responded to 5-HT and/or 8-OH-DPAT by coupling to several second messenger pathways. The 5-HT1A receptor inhibited, but did not stimulate, membrane adenylyl cyclase activity and whole cell cAMP accumulation in a dose-dependent manner (for 5-HT, IC50 = 146 +/- 27 and 55 +/- 12 nM, respectively). Activation of the receptor was associated with other signal transduction linkages: (i) a 40-50% increase in hydrolysis of inositol phosphates (for 5-HT, EC50 = 1.33 +/- 0.15 microM for 5-HT), (ii) a transient elevation of cytosolic Ca2+ levels (apparent at 1-100 microM 5-HT) which was not affected by chelation of extracellular Ca2+ by EGTA, and (iii) an augmentation of [3H]-arachidonic acid release pharmacologically with the calcium ionophore A23187 or by activation of endogenous thrombin or P2 purinergic receptors (for 5-HT, EC50 = 1.22 +/- 0.17 microM). This pathway may be an amplification mechanism for signaling in anatomic regions with high concentrations of several neuro-transmitters, hormones or autacoids, such as at neuronal junctions or near areas of platelet aggregation. All linkages were sensitive to pertussis toxin pre-treatment (IC50 approximately 0.5-0.6 ng/ml x 4.5 h for all pathways), suggesting the involvement of Gi protein(s) in these signal transduction pathways. Coupling to varied signal transduction pathways in a single cell system may be a common feature of receptors which classically inhibit adenylyl cyclase such as the 5-HT1A receptor.


Characterization of the interaction of the cervane alkaloid, imperialine, at muscarinic receptors in vitro. The action of the cervane alkaloid, imperialine, has been assessed at M1, M2 and M3 receptors in functional assays and at M1, M2, M3 and putative M4 sites in binding studies. In functional studies, imperialine acted as a selective surmountable antagonist at M2 receptors in guinea-pig isolated atria and uterus (-log KB = 7.7 and 7.4, respectively), in comparison to M1 receptors in canine isolated saphenous vein (-log KB = 6.9) or M3 receptors in a range of guinea-pig isolated smooth muscles including ileum, trachea, fundus, seminal vesicle or oesophagus (-log KB = 6.6-6.8). In rat aorta, the -log KB value at the M3 receptor (5.9) was slightly, but significantly, lower. In competition radioligand binding studies, imperialine was also selective toward to M2 sites in rat myocardium (-log Ki = 7.2) with respect to M1 and M3 sites (rat cerebral cortex, rat submaxillary gland; -log Ki = 6.1 and 5.7, respectively). However, it did not significantly discriminate between rat cardiac M2 sites and putative M4 sites in rabbit lung (-log Ki = 6.9). Imperialine resembles the alkaloid himbacine in terms of its pharmacological profile at muscarinic receptor subtypes in that it acts as an M2 selective antagonist with respect to M1 or M3 sites. It may also provide a second, commercially available, antagonist with which to discriminate between M1 and M4 receptors.


The extraneuronal transport mechanism for noradrenaline (uptake2) avidly transports 1-methyl-4-phenylpyridinium (MPP+). The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake2) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake2 has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake2 in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP+). Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake2. The initial rate of 3H-noradrenaline transport [kin = 0.58 microliter/(mg protein.min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (kO-methylation = 0.54 min-1). The neurotoxin MPP+ was found to be a good substrate of uptake2. Initial rates of specific 3H-MPP+ transport into Caki-1 cells were saturable, the Km being 24 micromol/l and the Vmax being 420 pmol/(mg protein.min). The rate constant of specific inward transport was 34 times higher [19.6 microliters/(mg protein.min)] than that of 3H-noradrenaline. The ratio specific over non-specific transport was considerably higher for 3H-MPP+ (12.6) than for 3H-noradrenaline (3.0). 3H-MPP+ transport into Caki-1 cells was inhibited by various inhibitors of uptake2. The highly significant positive correlation (p less than 0.001, r = 0.986, n = 7) between the IC50's for the inhibition of the transport of 3H-noradrenaline and 3H-MPP+, respectively, proves the hypothesis that MPP+ enters Caki-1 cells via uptake2. 3H-MPP+ is taken up via uptake2 not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake2.(ABSTRACT TRUNCATED AT 250 WORDS)


Extraneuronal uptake of noradrenaline in rabbit dental pulp: evidence of identity with uptake1. The extraneuronal removal and disposition of noradrenaline in rabbit dental pulp was examined in view of earlier evidence that the tissue possessed an extraneuronal uptake process resembling neuronal uptake1. Pulp, which had been depleted of sympathetic nerves by homolateral superior cervical ganglionectomy, was incubated in vitro with 3H-noradrenaline in low concentrations (0.025 or 0.18 mumol/l). When the metabolising enzymes (monoamine oxidase, catechol-O-methyl transferase) were active, 3H retention by the denervated pulp, as indicated by the 3H content after the tissue had been washed for 30 min following incubation with 3H-noradrenaline, was less than 3% of that of the innervated pulp. When the enzymes were inhibited, retention rose to approximately 30% of that of the innervated pulp. Analysis of the time course of the 3H efflux indicated that the 3H-noradrenaline in the denervated pulp had accumulated in a single compartment characterised by a t1/2 for efflux of several hours. Accumulation did not occur under Na(+)-free conditions, and was inhibited by desipramine (IC50 less than 0.03 mumol/l) and by substrates of neuronal uptake1. Mean IC50 values of the latter were very similar to those for inhibition of neuronal uptake1 and comprised (in mumol/l): (+)amphetamine (0.29), dopamine (0.31), tyramine (0.39), (-)noradrenaline (0.70), (-)adrenaline (1.50), 5-hydroxytryptamine (20) and bretylium (35). Uptake2 inhibitors were less active (O-methyl isoprenaline, IC50 = 60 mumol/l) than uptake1 inhibitors, or were without inhibitory effects at the concentrations tested (hydrocortisone, 210 mumol/l; 2-methoxy oestrone, 10 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)


Stable adenine nucleotides inhibit [3H]-noradrenaline release in rabbit brain cortex slices by direct action at presynaptic adenosine A1-receptors. Effects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz. Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADP beta S, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine greater than ATP approximately ATP gamma S approximately beta,gamma-imido-ATP approximately ADP greater than beta,gamma-methylene-ATP. AMP 30 mumol/l and AMPS 30 mumol/l were approximately equieffective with 30 mumol/l of adenosine and ATP gamma S, and ADP beta S 30 mumol/l was approximately equieffective with 30 mumol/l of ADP. alpha,beta-Methylene-ADP, 2-methylthio-ATP, UTP and GTP gamma S did not change the evoked overflow of tritium. alpha,beta-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to alpha,beta-methylene-ATP or when the slices were stimulated by 30 pulses, 10 Hz. Neither adenosine deaminase (100 U/l) nor the blocker of 5'-nucleotidase, alpha,beta-methylene-ADP (10 mumol/l), attenuated the inhibition caused by ATP, ATP gamma S and beta,gamma-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP to the right by very similar degrees. 8-(p-Sulphophenyl)-theophylline (30 and 300 mumol/l) also markedly antagonized the inhibition produced by ATP gamma S. alpha,beta-Methylene-ATP (10 and 30 mumol/l) and suramin (100 mumol/l) did not modify the effects of adenosine, ATP gamma S and beta,gamma-methylene-ATP. It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A1 subtype of the P1-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP can be potent P1 agonists. No evidence was found for presynaptic P2x-, P2y- or P3-purinoceptors.


Lidocaine has a narrow antiarrhythmic dose range against ventricular arrhythmias induced by programmed electrical stimulation in conscious postinfarction dogs. The aim of the present study was to investigate the dose-dependent antiarrhythmic efficacy of lidocaine against electrically induced tachycardias in conscious, chronically instrumented postinfarction dogs. Programmed electrical stimulation (PES) was performed in 16 dogs 8 to 21 days after a 4 h occlusion of the left anterior descending coronary artery (LAD). Infusion of saline in 8 control animals with sustained ventricular tachycardia (SVT) inducible at baseline did not affect subsequent inducibility. In the treatment group 7 of 8 animals responded with SVT and one exhibited ventricular fibrillation at baseline. After an initial bolus of 1 mg/kg lidocaine intravenously (i.v.), the drug was infused at infusion rates of 40, 80 and 120 micrograms/kg/min (i.v.). During 80 micrograms/kg/min lidocaine (mean plasma level 3.5 micrograms/ml) 7 out of 8 animals displayed an antiarrhythmic response; both the lower and the higher infusion rate were associated with a smaller antiarrhythmic efficacy (3 of 8 animals responded to 40 micrograms/kg/min and 4 of 8 to 120 micrograms/kg/min). Licocaine did not affect ventricular refractory periods, but induced an increase in intraventricular conduction time at all infusion rates, from 66.2 ms at baseline to 67.7 ms (p less than 0.05), 67.7 ms (p less than 0.05), 70.0 ms (p less than 0.01) respectively. In conclusion the present study demonstrates that lidocaine is of considerable value in the management of PES-induced ventricular arrhythmias in the postinfarction phase. However there is only a small optimal therapeutic plasma level range, where lidocaine exhibits its antiarrhythmic efficacy against this type of arrhythmia; this makes a carefully titration of the drug necessary both in the experimental and in the clinical setting.


Blood pressure and gastric motor responses to bradykinin and hydrochloric acid injected into somatic or visceral tissues. Both visceral and somatic nociceptive stimuli elicit reflex changes in blood pressure and gastric motor activity, but the exact type of response varies with the type of nociceptive stimulus and its site of application. Therefore, the present study compared the effects of visceral (i.p. or i.a.) and somatic (s.c.) administration of bradykinin and HCl on both mean arterial blood pressure (MAP) and intragastric pressure in anaesthetized rats. The nervous pathways mediating these responses were investigated by surgical or pharmacological inhibition of the possible reflex arcs. Bradykinin (i.a.--into the aortic arch, i.p., and s.c.), and HCl (i.p. and s.c.), elicited a fall in MAP followed by a transient increase. Intragastric pressure decreased in response to administration of these chemicals. Acute coeliac ganglionectomy reduced the gastric relaxations in response to both bradykinin and HCl, whereas vagotomy reduced only the gastric relaxations induced by HCl. Neither lesion influenced the changes in MAP after either chemical. Ablation of small diameter afferents by capsaicin or chemical sympathectomy by guanethidine reduced the changes in MAP after both chemicals, except that which occurred after i.a. injection of bradykinin. The secondary increase in MAP after i.a. and i.p. administration of algesics was increased after guanethidine. Both pretreatments reduced gastric relaxations in response to either chemical. Pretreatment of the rats with the bradykinin antagonist Hoe-140 reduced the responses to bradykinin but not to HCl. The results show that both visceral and somatic administration of painful chemicals elicit reflex falls in MAP and intragastric pressure.(ABSTRACT TRUNCATED AT 250 WORDS)


Hydroxysteroid sulfotransferase and a specific UDP-glucuronosyltransferase are involved in the metabolism of digitoxin in man. In vitro experiments were performed with cytosolic and microsomal fractions of human liver specimens in order to investigate which enzyme forms of sulfotransferase (ST) and UDP-glucurosyltransferase (GT) are involved in the metabolism of digitoxin (dt-3) and/or its cleavage products. It was found that the cytosolic STs preferentially react with digitoxigenin (dt-0) whereas microsomal GTs conjugate digitoxigenin-monodigitoxoside (dt-1) and in traces the bisdigitoxoside (dt-2). Dt-3 and dt-0 cannot be glucuronidated. By separation of different sulfotransferases it was found that the hydroxysteroid-ST is responsible for dt-0 and 3-epidigitoxigenin (epi-dt-0) sulfation. The hydroxysteroid-ST could be purified and characterized (apparent Km and Vmax for dt-0 sulfation: approx. 17 mumol/l and 2.7 nmol/min mg protein, respectively). Of various model substrates and endogenous compounds (steroids, bilirubin) none caused a competitive inhibition of the microsomal dt-1 glucuronidation except dt-2 and dt-3. Therefore it can be supposed that a new GT form catalyses this reaction. It is characterized by an extraordinarily high affinity towards dt-1 with Km values ranging between 0.7 and 27 mumol/l.


Reduction of postischemic leukocyte-endothelium interaction by adenosine via A2 receptor. The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg-1 min-1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p less than 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces post-ischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.


[Intentional hypotension therapy induced by nitroglycerin and the changes in the intracranial pressure for acute hemorrhagic intracranial lesions]. It is reported that nitroglycerin (NTG) induces the elevation of intracranial pressure (ICP). Because of it, the use of NTG in patients exhibiting the increased ICP especially in the neurosurgical field is thought to be avoided. However, these reported cases dealt with normal patients. Few cases under the condition of exactly increased intracranial pressure were studied. Our 31 patients of the hemorrhagic intracranial lesion (subarachnoid hemorrhage and hypertensive intracranial hemorrhage) at the acute stage were treated by NTG infusion as an antihypertensive drug, and the ICP was measured using the epidural pressure transducer. The 31 patients were divided into three groups. Group 1 consists of the 14 patients exhibiting normal ICP (< 15mmHg). Group 2 consists of 9 patients showing elevated ICP (15mmHg < or =). Group 3 consists of 8 patients treated with glycerol before the NTG infusion. Group 1 demonstrates a statistically significant elevation of ICP corresponding with the blood pressure depression. Group 2 showed no significant elevation of it. Group 3 disclosed no remarkable elevation of it both in the elevated ICP cases and the normal ICP cases. As mentioned above, the intravenous NTG caused a remarkable increase of ICP in the normal compliance of the intracranial contents and no elevation of ICP in the poor compliance. We conclude that NTG can be used for blood pressure control at an acute stage even in hemorrhagic intracranial lesion as anti-depressor.


[Evaluation of cerebral blood circulation using fast magnetic resonance imaging (turbo-FLASH)]. In its initial stage of development, poor time resolution was supposed to be inevitable for magnetic resonance imaging (MRI). The development of the gradient echo technique, however, has made an opening for a breakthrough to a fast MRI technique. However, conventional gradient echo technique is not fast enough to disclose the cerebral blood circulation. A new technique (turbo-FLASH), which combines gradient echo and preparation pulse, has opened the door to a true dynamic (high time resolution) MRI. The authors performed a basic study of this new fast imaging technique (Turbo-FLASH) for the evaluation of cerebral blood circulation. Using this dynamic MRI, the conventional Gd-DTPA administration (0.1mmol/kg, intravenous manual administration) has produced sufficient contrast to reveal a difference of the extent of enhancement between normal gray matter and white matter. In patients of cerebral infarction, the infarcted brain does not show any significant increase of signal intensity after the administration of contrast medium, whereas the normal brain shows a sharp increase of signal intensity after the administration of contrast medium. Indeed, many puzzling questions remain to be solved for clinical application of this new dynamic MRI. For example, the optimal quantity of contrast medium and optimal parameters for this sequence (T1 weighted or T2 weighted?) are still unknown. But, in any case, this dynamic MRI is expected to be a clinically powerful tool for the evaluation of cerebral blood flow.


[Basilar artery occlusion therapy for giant aneurysm: hemodynamic analysis by hydraulic vascular model]. Therapeutic occlusion of the basilar artery has been one of the alternative treatments for surgically or intravascularly inaccessible basilar bifurcation giant aneurysms. However, several problems have been reported, such as incomplete thrombosis of the aneurysms, their growth or rupture, and cerebral embolism originating from their cavities. Since hemodynamic changes after occlusion therapy are suspected to be responsible for these phenomena, they were investigated by a hydraulic vascular model. A hydraulic vascular model of the vertebrobasilar artery was constructed with silicone and glass tubes. A glass-made sphere of 2.5 cm in diameter was attached to the model and was regarded as a basilar head aneurysm. A 40% glycerol solution at 25 degrees C was found to be of similar viscosity and specific gravity to those of human whole blood at 37 degrees C and was perfused in the model. A device to measure intra-aneurysmal clearance was made from a stable luminous source and a Cds photocell. Good correlation was found between the output and an intra-aneurysmal dye concentration. The dye was injected into the aneurysm and its half-life was calculated from clearance curves. It was then regarded as an index of stagnation in an aneurysmal cavity. The flow volumes were estimated as: 60ml/min to the territory of one posterior cerebral artery (PCA) and 80ml/min to the cerebellum and the brain stem. Half-life was recorded in the following conditions: 120ml/min of flow in the basilar artery (BA) into bilateral PCAs stimulating the condition before BA occlusion, and various flow values (60ml/min to 10ml/min) of P1 segment simulating the conditions after BA occlusion distal to the superior cerebellar artery.(ABSTRACT TRUNCATED AT 250 WORDS)


[Ischemic complications following parent artery occlusion with detachable balloons in the treatment of large carotid aneurysms: effectiveness of 99mTc-HMPAO SPECT and importance of postoperative management]. Three patients with large carotid aneurysms were treated by parent artery occlusion with detachable balloons. Of these, 2 had intracavernous carotid artery aneurysms and one had a carotid-ophthalmic aneurysm. All patients underwent a formal balloon occlusion test and tolerated it well. One patient with a carotid-ophthalmic aneurysm, however, developed postoperative ischemic effects. This case was that of a 55 year-old female with right visual disturbance. Her middle cerebral artery flow had been compromised during test occlusion under induced hypotension. Although initially intact after balloon occlusion, she was found to be hemiplegic 24 hours later. She subsequently developed multiple small infarcts which were shown on MRI and which corresponded exactly to the previously demonstrated region of decreased flow during testing. This ischemic complication is possibly due to hypoperfusion, but embolism from the thrombosed aneurysm can not be denied. To prevent these complications, an extracranial-intracranial artery anastomosis prior to parent artery occlusion should be considered if the preoperative 99mTc-HMPAO SPECT shows a compromised cerebral blood flow. In addition to this, postoperative anticoagulant therapy should be given even if test occlusion was well tolerated clinically.


[Cyst formation following local chemotherapy of malignant brain tumor: a clinicopathological study of two cases]. This paper reports clinicopathological findings concerning an enlarged bulky cyst and the tumor cavity following local administration of an anticancer agent combined with radiotherapy in two patients suffering from malignant glioma. Case 1: This 69 year-old man who had been diagnosed as having glioblastoma in the right parietal lobe had received local chemotherapy after the first operation. Simultaneously radiotherapy of 69 Gy in total dose was performed. At the second operation for the tumor, cyst formation was clinically confirmed and necrotomy as well as evacuation of the large cyst was performed after adjuvant therapy. The patient died at a time ten months after the first surgical operation. Case 2: This is the case of a 48 year-old man who was diagnosed as having gemistocytic astrocytoma in the left frontal lobe. The first surgical operation was performed and was followed by local chemotherapy as well as radiotherapy (total dose of 90 Gy in two sessions). The second surgical operation of the recurrent tumor, with necrotomy and evacuation of the large cyst were performed after adjuvant therapy. The patient expired at a time sixty-five months after the first surgical operation. Relevant to the chemotherapy, adriamycin (ADM) (0.5mg) and methotrexate (MTX) (1mg) were administered through the Ommaya's reservoir into the tumor bed at craniotomy. The usual doses of ADM and MTX amounted to 5.0mg respectively. Through conventional CT and MRI, formation of a cyst including abundant membranous debris or septi was identified in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)


[A case of common carotid artery stenosis due to hanging]. We report a case of a 50-year-old woman with traumatic dissection of the right common carotid artery secondary to attempted suicidal hanging. Initial examination showed marks of strangulation on the neck, facial edema, and multiple conjunctival petechiae accompanied by difficulty in breathing and loss of consciousness. She gradually recovered except for her left upper limb weakness. Rehabilitation, with a diagnosis of left brachial plexus injury, was started. Two years later, she occasionally felt numbness of the left upper extremity. On her second admission, a bruit was heard on the right side of her neck. Neurological examination did not reveal any change. CT scan showed atrophic changes. Angiography demonstrated approximately 80% stenosis of the right common carotid artery which was thought to be responsible for the attacks of numbness. Right carotid endarterectomy was performed. Histological findings revealed dissection of the medial layer of the arterial wall. Complaints of numbness disappeared postoperatively, and angiography showed good patency at a follow-up examination. We conclude that the stenosis of the common carotid artery was not due to atherosclerosis but due to the dissection as a result of hanging.


[A case of subfrontal schwannoma].

[A case of subfrontal schwannoma]. We encountered a rare case of subfrontal schwannoma. A 55-year-old woman had received resection of a left frontal tumor because of hyposmia, at the age of 28 years. On June 10, 1989, she was admitted with the chief complaint of progressive contraction of visual field. Neurologic examination revealed anosmia, impaired vision and concentric contraction of visual field. Fundoscopy showed optic atrophy. CT examination demonstrated a calcified mass of mixed density which was occupying her nasal cavity, ethmoid sinus and anterior skull base. The lesion was enhanced with contrast medium. MRI clearly depicted the extension of the lesion and a low signal intensity area in the left frontal lobe as a postoperative scar. Angiography showed hypovascularity. The tumor was totally removed by bifrontal craniotomy on August 22, 1989. Infiltration into the brain or compression of the optic nerve was not detected. The dura on the cranial base side was damaged and lost by infiltration of the tumor, normal olfactory bulb was not able to be identified, and the cribriform plate was broken. The anterior skull base was reconstituted by covering the dural defect with cadaveric dura and the bony defect with a pericranium. HE staining showed Antoni A&B types of schwannoma. Postoperative course was uneventful. In this case, it is most likely that a remnant of the tumor resected when she was 28 years old had developed subfrontal schwannoma a long time after the operation, although the histological type at that time was unknown. It is also possible that a primary tumor in the nasal cavity or paranasal sinus may have extended into the cranium.(ABSTRACT TRUNCATED AT 250 WORDS)


[Gliosarcoma associated with von Recklinghausen's disease: a case report]. A very rare case of gliosarcoma with von Recklinghausen's disease is presented. A 51-year-old man was admitted to our hospital in March 1990, because of a 2-month history of personality change and left hemiparesis. Multiple neurofibroma over his whole body with many café au lait spots had been present since early childhood. His mother, brothers and children also had café au lait spots. Neurological examination on admission revealed memory disturbance, left homonymous hemianopsia and left hemiparesis. CT scan showed a large lobular lesion in the right temporoparietal region. The medial hypodense part was sharply demarcated by a ring-like enhancement, while the lateral isodense part was homogeneously enhanced. MRI showed a sharply demarcated high intensity lesion with Gd-EDTA enhancement corresponding to the enhanced area on CT. Faint staining on angiography revealed that the blood supply to the tumor was predominantly dural. At surgery, the tumor was solid and highly vascular, and adhered tightly to the dura. The superficial part of the tumor was well demarcated from the brain tissue, but the demarcation was obscure in the deeper part. Histological findings showed two clearly defined neoplastic components: a gliomatous component that was stained for GFAP, and a sarcomatous component that had spindle-shaped nuclei and eosinophilic fibers. The patient underwent radiotherapy after surgery, but the tumor soon recurred. A second operation was performed, but the tumor had infiltrated into the scalp and he died 10 months after the first operation. This combination is very rare and has not been reported previously.


[A case of cystic optic glioma involving chiasma and bilateral posterior optic pathway]. A case of cystic optic glioma involving chiasma and bilateral posterior optic pathway was reported. A 26-year-old male was admitted to our hospital complaining of dysarthria and left hemiparesis. CT, MRI revealed a cystic tumor at the right basal ganglia to midbrain, a calcified one at the bilateral optic tract and left temporal to thalamic region, and a small one at the chiasma. Radiotherapy and chemotherapy were performed because anaplastic astrocytoma was suspected after stereotactic biopsy of the tumor at the right basal ganglia. The subsequent MRI showed continuity among the above three lesions to be well defined. About 2 years later, however, enlargement of the cyst, tumor invasion beyond the optic pathway and growth of the chiasmal lesion were noted, and direct surgery to the chiasmal lesion was performed. The chiasma was swollen and grayish soft tumor tissue was partly resected after aspiration of the intrachiasmal cyst. The definitive pathological diagnosis was pilocytic astrocytoma. This case was designated as a peculiar optic glioma in the following respects; the patient was an adult man suffering from dysarthria and left hemiparesis, the tumor involved not only the chiasma and the bilateral optic tract, but also the outside optic pathway and was accompanied by a large cyst.


[Two cases of failure of a programmable pressure valve]. Two cases of a programmable pressure valve trouble are reported. Case #1 was that of a 49-year-old male who had received a ventriculoperitoneal shunt (VPS) using a Sophy programmable pressure valve (Model S3) after the removal of the fourth ventricle ependymoma. After the VPS, the patient went through several episodes of complaining of headache due to the spontaneous movement of the rotor from the high pressure position to the medium or low pressure position. The symptom had disappeared after exchanging the valve for a new one and by setting the valve pressure at high. Examination of the removed valve revealed that the bending-degree of the spring tip was insufficient, and that the opening pressures at the medium and high pressure positions were both lower than the desired range. Case #2 was that of a 62-year-old female who had received a VPS for normal pressure hydrocephalus using a Sophy programmable pressure valve (Model S3) with the valve pressure set a high. However, her symptoms did not improve, so an attempt was made to change the valve pressure from the high pressure position to the medium pressure position with a ring magnet. But the rotor could not be moved. The valve was exchanged and the valve pressure was set at medium, and the symptoms of the patient improved postoperatively. Examination of the removed valve revealed that the movement of the rotor with a ring magnet was not able to be carried out smoothly.(ABSTRACT TRUNCATED AT 250 WORDS)


[Primary intracranial melanoma: a case report]. A rare case of primary intracranial melanoma is presented in a 34-year-old man with initial symptoms of persistent headache. In magnetic resonance imaging(MRI), this case had all the characteristic findings of intracranial melanoma which had been reported previously. In 123I-iodoamphetamine-single photon emission CT (123I-IMP-SPECT), abnormal accumulation of 123I-IMP was recognized in early and late phase imaging, which was very specific to the lesion. This is the first report of 123I-IMP-SPECT performed on a primary intracranial melanoma. Tumor mass originated from pia mater was surgically resected, but the dissemination of tumor cells was recognized macroscopically. Pathological examination of the specimen showed very little malignant changes of melanoma cells, which was in contrast to the previous reports. Although, no standard chemotherapy of the primary intracranial melanoma has been established, DAV therapy to the dissemination of tumor cells into the subarachnoid space, and intravenous administration of interferon-beta were performed in this case. Methods of differential diagnosis and treatments of primary intracranial melanoma are reviewed and discussed.


[Anterior sacral meningocele associated with tethered cord syndrome]. A case of anterior sacral meningocele associated with tethered cord syndrome is reported. A 5-year-old boy was admitted for urinary and fecal incontinence which had persisted since his birth. Abdominal MRI and sacral CT showed a presacral cystic mass communicating with the spinal dural sac through a neck traversing a sacral bony defect and tethered spinal conus. Neurological examination showed that the patient had a neurogenic bladder, atrophy of the legs and anesthesia below the S3 level. Operative finding revealed a thickened filum terminale attached to the wall of the presacral meningocele. Agenesis of the nerve root below the S2 level was identified, and no neural elements entered into the sac. Untethering was performed. Postoperative course was uneventful and the patient is doing well with his neurogenic bladder gradually improving.


On the significance of subterritories in the "accumbens" part of the rat ventral striatum. Although many workers have appreciated the striking cytologic and neurochemical similarities of neostriatum, accumbens and olfactory tubercle, a compelling case for regarding these areas as territories in a striatal complex awaited the arguments made by Heimer and his colleagues based on their investigations of connections. A number of recent papers support this viewpoint and extend it with the characterization of three accumbal subterritories: core, shell and rostral pole. The case for separate classifications of systems traversing the accumbens has become more compelling with each study that demonstrates connectional, cytoarchitectural and neurochemical specificity conforming to the boundaries separating the core and its downstream targets from the shell and its projection fields. Furthermore, its apparent composite of core-like and shell-like characteristics distinguishes the rostral pole as yet another unique subterritory. Differences in compartmental organization distinguish the accumbens and neostriatum. The available data are consistent with the periventricular and rostrolateral enkephalin-rich zones being ventralmost parts of the neostriatal patch and matrix compartments, respectively. The accumbal cell cluster compartment, on the other hand, appears to be a separate entity, with connectional and neurochemical features that are dissimilar to both patch and matrix of neostriatum. Boundaries between the accumbens and caudate-putamen remain elusive, and the point of view that such boundaries do not exist but, rather, are represented by "transition zones" must to a large degree reflect the reality. Likewise, it is important to acknowledge that the boundaries between accumbal subterritories are not necessarily distinct or observed faithfully by all of the afferent systems. "Transition zones" appear to be particularly significant organizational features in rostral and lateral parts of the accumbens. Interestingly, histochemically distinct cell clusters tend to be numerous in boundary regions between adjacent territories and subterritories. The predominant organizational pattern appears to be one in which the core, shell and rostral pole engage different forebrain systems that possibly subserve entirely different functions mediated by distantly related mechanisms. In this regard, it is of paramount interest that the processing of information conveyed to the accumbens by diverse cortical and subcortical inputs occurs within distinct and perhaps very different dopaminergic environments in the core, shell and rostral pole (e.g., see Refs 24, 34, 90, 110).


Pharmacological stimulation reveals recombinant human nerve growth factor-induced increases of in vivo hippocampal cholinergic function measured in rats with partial fimbrial transections. The present study determined the effects of chronic recombinant human nerve growth factor administration [1 microgram given intracerebroventricularly q.i.d. (every other day) for three weeks] on in vivo hippocampal cholinergic function in adult rats with unilateral partial fimbrial transections. Partial fimbrial transections did not significantly alter the levels of endogenous acetylcholine or [2H4]acetylcholine in the hippocampus due to functional compensation by surviving cholinergic terminals. In animals chronically treated with nerve growth factor, the levels of endogenous choline, endogenous acetylcholine, [2H4]choline and [2H4]acetylcholine accumulated in the hippocampus on the lesioned side were not significantly different from those on the contralateral unlesioned side or from values measured in animals treated with cytochrome c, a control protein. However, changes in cholinergic parameters induced by the partial lesions or recombinant human nerve growth factor treatment became manifest when animals were challenged using pharmacological agents such as pentylenetetrazole or pilocarpine given after lithium chloride pretreatment. First, in nerve growth factor-treated animals administered the general stimulant pentylenetetrazole (10 mg/kg) 2 min prior to measuring in vivo cholinergic parameters, we observed a significant increase in the hippocampal content of [2H4]choline in both lesioned and unlesioned hippocampi. The magnitude of the increase was significantly higher on the lesioned compared to the unlesioned side. Although chronic recombinant human nerve growth factor treatment induced increases of hippocampal [2H4]choline levels, there were no concomitant increases in the level of [2H4]acetylcholine. Second, in nerve growth factor-treated animals administered lithium chloride (3 mmol/kg) 20 h prior to pilocarpine (30 mg/kg), we observed a significant enhancement of the content of endogenous acetylcholine in the hippocampus of the lesioned side. Partial fimbrial transections also reduced in vitro cholinergic parameters reflecting endogenous acetylcholine levels in hippocampal slices. The content of endogenous acetylcholine in the slices was decreased by approximately 50% and chronic nerve growth factor treatment significantly elevated this value to approximately non-lesioned control values. Similarly, reductions in spontaneous and veratridine-evoked release of endogenous acetylcholine induced by partial fimbrial transections were counteracted by recombinant human nerve growth factor treatment. These findings demonstrate that chronic recombinant human nerve growth factor treatment effectively enhances the in vivo and in vitro synthesis, storage and release of endogenous acetylcholine. The results from the in vivo studies suggest that recombinant human nerve growth factor-induced differences in functional performance of hippocampal neurons may only be manifest during behavioral and/or pharmacological stimulation.


State-dependent and state-independent effects of thyrotropin-releasing hormone on medial septum neuronal activity in the brain slices of waking and hibernating ground squirrels. Effects of thyrotropin-releasing hormone on spontaneous activity and responses to medial forebrain bundle stimulation were tested in the units of the medial septum-diagonal band complex in slices taken from the brain of hibernating and waking ground squirrels. Administration of thyrotropin-releasing hormone (0.1 microM) into the flow of incubating medium increased the frequency of spontaneous activity of all the medial septum-diagonal band complex neurons in hibernating ground squirrels and of the majority of neurons in the waking ground squirrels. However, in the septal slices of hibernating ground squirrels this increase was significantly more pronounced. In addition, the neuropeptide slightly increased the frequency of bursts in the majority of cells with rhythmic burst activity. The excitatory influence of thyrotropin-releasing hormone on the units was preserved in conditions of synaptic blockade. In neurons from other structures (lateral septum, medial preoptic area, hippocampus) in the brain slices of both hibernating and waking ground squirrels, thyrotropin-releasing hormone did not usually affect the level of spontaneous discharges. When studying the responses of the medial septum-diagonal band complex neurons to electrical stimulation of medial forebrain bundle it was found that application of thyrotropin-releasing hormone (0.1 microM) led to the disappearance of responses in 50 and 44% of units in the hibernating and waking ground squirrels, respectively; in the rest of the neurons a disturbance of stability and probability of responses was observed. The existence of a modulatory thyrotropin-releasing hormone system which participates post-, and, probably, presynaptically in the regulation of the medial septum-diagonal band complex neuronal activity is suggested. The role of thyrotropin-releasing hormone and of medial septum-diagonal band complex in the neural control of hibernation/euthermic waking cycle is discussed.


Fos-like expression and nuclear size in osmotically stimulated supraoptic nucleus neurons. This study has analysed by immunocytochemistry the pattern of expression of Fos-related proteins, as well as variations in nuclear size, after the osmotically induced activation of supraoptic nucleus neurons of the rat. In control rats most supraoptic nucleus neurons were Fos-like negative. After acute and chronic dehydration by salt-loading, the number of Fos-like positive neurons increased dramatically. The level of Fos-like immunoreactivity was higher in chronically stimulated rats, and also the neurons of the ventral region of the supraoptic nucleus were more intensely stained than those of the dorsal region. The karyometric analysis was made on electron micrographs. The mean nuclear profile area showed a significant increase in dehydrated rats with respect to the controls (73 +/- 16 microns 2 in those dehydrated for six days vs 54 +/- 13 in controls, mean +/- S.D.). However, no significant differences in this parameter were found when one-day and six-day dehydrated groups were compared. The invagination factor of the nuclear membrane, a nuclear shape indicator, decreased significantly in dehydrated rats, indicating a tendency towards spherical nuclei. It is noteworthy that the nuclear profile perimeter was constant, about 32 microns, in control and osmotically simulated rats. The higher nuclear accumulation of Fos-related antigens after six days of dehydration suggests that in chronically stimulated supraoptic nucleus neurons there is a sustained induction of cell-specific genes. Moreover, the transcription rate of the target genes containing the consensus DNA sequence TGAC/GTCA or c-AMP responsive elements recognition sites may depend upon the nuclear concentration of Fos-related antigens in supraoptic nucleus neurons. Our results also suggest that the initial Fos-related antigen expression and nuclear size increase are triggered concomitantly in supraoptic nucleus neurons after a short period of osmotic stimulation. On the other hand, we propose that nuclear envelope invaginations represent a reservoir of nuclear membrane which allows dynamic changes in nuclear size and shape depending on the metabolic status of the supraoptic nucleus neurons.


Lesion and electrophysiological studies on the hypothalamic afferent pathway of the milk ejection reflex in the rat. The afferent pathway of the milk ejection reflex in the hypothalamus was investigated with lesion and electrophysiological methods in anesthetized lactating rats. Destruction of the central region of the mid-hypothalamus (n = 12) blocked milk ejections induced by suckling, while that of the lateral region (n = 7) had no effect. In an electrophysiological study, extracellular recordings of neurons antidromically activated by electrical stimulation of the supraoptic nucleus were obtained from the ipsilateral hypothalamus caudal to the paraventricular nucleus (n = 84). Thirty-nine neurons were examined to see whether their firing activities changed during the milk ejection reflex. A group of 13 neurons were found to show changes in their activities prior to the reflex milk ejection; the neurons displayed a brief high-frequency burst of spikes before each milk ejection in the same manner as oxytocin neurons, and none of them antidromically responded to electrical stimulation of the neurohypophysis. The bursting neurons were recorded from the dorsomedial hypothalamic nucleus (n = 6), the region just lateral to that nucleus (n = 3) and the posterior hypothalamus (n = 4). The locations were included in a region whose destruction blocked the milk ejection reflex. These results indicate that the afferent pathway of the milk ejection reflex in the rat runs through the medial portion of the hypothalamus posterior to the paraventricular nucleus and that this region contains neurons which relay the input to the oxytocin neurons projecting in the neurohypophysis.


5-Hydroxytryptamine-induced sensitization and activation of peripheral fibres in the neonatal rat are mediated via different 5-hydroxytryptamine-receptors. The effects of 5-hydroxytryptamine on peripheral nociceptive fibres were studied in an in vitro preparation of the neonatal rat spinal cord with attached tail. The activation of peripheral fibres in the tail by noxious stimuli (bradykinin, capsaicin, heat) was recorded as a depolarization of a ventral root in the lumbar region of the spinal cord (L3-L5). Responses evoked by brief applications of submaximal or threshold concentrations of bradykinin or capsaicin to the tail were enhanced by 5-hydroxytryptamine and the 5-hydroxytryptamine1C/5-hydroxytryptamine2-receptor agonist alpha-methyl-5-hydroxtryptamine but not by the 5-hydroxytryptamine3-receptor agonist 2-methyl-5-hydroxytryptamine or the 5-hydroxytryptamine1-receptor agonist 5-carboxamidotryptamine. Sensitization induced by 5-hydroxytryptamine and alpha-methyl-5-hydroxytryptamine was blocked by the selective 5-hydroxytryptamine2-receptor antagonist ketanserin. Neither the 5-hydroxytryptamine3/5-hydroxytryptamine4-receptor antagonist ICS 205-930 nor the 5-hydroxytryptamine1/5-hydroxytryptamine2-receptor antagonist methiothepin blocked the 5-hydroxytryptamine-induced sensitization. The responses evoked by submaximal thermal stimuli were also enhanced following the sensitization of peripheral nociceptors with 5-hydroxytryptamine or alpha-methyl-5-hydroxytryptamine. The alpha-methyl-5-hydroxytryptamine-induced enhancement of thermal responses was reduced by ketanserin. 5-Hydroxytryptamine did not evoke a ventral root response unless peripheral fibres were sensitized with threshold concentrations of bradykinin or capsaicin. This effect was mimicked under the same conditions by 5-carboxamidotryptamine but not by alpha-methyl-5-hydroxytryptamine or 2-methyl-5-hydroxytryptamine. The excitatory effect of 5-hydroxytryptamine was blocked by methiothepin but not by ICS 205-930 or ketanserin. Neither 5-hydroxytryptamine-induced sensitization nor 5-hydroxytryptamine-evoked activation of peripheral fibres was blocked by indomethacin. These data indicate that two types of receptor are involved in the peripheral actions of 5-hydroxytryptamine in nociception. 5-Hydroxytryptamine-induced sensitization involves a 5-hydroxytryptamine2-receptor, whereas 5-hydroxytryptamine-evoked excitation involves a 5-hydroxytryptamine1-like-receptor.


Measurements of intracellular calcium in sensory neurons of adult and old rats. Cytosolic free calcium ([Ca2+]i) was measured using fluorescent digital imaging microscopy in rat dorsal root ganglion neurons isolated from animals of two age groups (adult: seven months; and old: 30 months). Neurons were enzymatically isolated and maintained in primary culture for 14 days. Cultured neurons were loaded with the fluorescent dye, Fura-2. The spatial distribution of resting [Ca2+]i was even in both adult and old rats, but the value of cytoplasmic free calcium in old neurons was significantly higher (207 +/- 37 nmol/l vs 96 +/- 23 nmol/l) in comparison with adult ones. Depolarization with 50 mmol/l K+ produced a rapid increase in [Ca2+]i in all neurons, but the values of depolarization-induced increase of [Ca2+]i in old neurons were significantly lower (423 +/- 54 nmol/l) compared with cells isolated from adult rats (1011 +/- 91 nmol/l). The time of the complete restoration of [Ca2+]i to the resting level was 10-times longer in old neurons. The caffeine-induced rise of intracellular calcium was somewhat higher in neurons from old animals, and its restoration to normal level was delayed. The findings indicate a substantial alteration of the mechanisms of regulation of intracellular calcium homeostasis with neuronal ageing.


Long-term reduction of vasopressin excretion induced by the central injection of an immunoconjugate (antibody to vasopressin linked to ricin A chain). We have previously demonstrated that vasopressin-producing neurons are the target of monoclonal antibodies to vasopressin microinjected into the brain tissue. At the same time, this central microinjection of vasopressin-monoclonal antibody into the supraoptic nuclei produced hydro-osmotic disorders mimicking the effects of a central diabetes insipidus. In order to investigate the increase in both duration and amplitude of the biological effects seen after the injection of vasopressin-monoclonal antibody, an immunoconjugate was constructed with the vasopressin-monoclonal antibody IgG1k isotype and the cytotoxic part of the ricin molecule, the ricin A chain. The biological parameters, such as diuresis and urine osmolality which are directly regulated by vasopressin, and vasopressin excretion, were measured after the central injection of this immunotoxin/immunoconjugate. The consequences of immunotoxin injection were also studied when immunotoxin was co-injected with monensin (50 nM) which has been shown to decrease the intracellular degradation of immunotoxin, and plasma complement, which has been shown to increase the neuronal uptake of immunotoxin. Single injection of immunotoxin near the hypothalamic supraoptic nuclei significantly increased diuresis and decreased vasopressin excretion. However, these effects were only transient and disappeared 24 h later. Four successive injections of immunotoxin (one per day) with monensin induced a decrease of vasopressin excretion which was still observed after a resting period of four days after the fourth injection. The long-term reduction of vasopressin excretion was induced in rats receiving four successive injections of a mixture consisting of immunotoxin with monensin and plasma complement. In such experiments, the vasopressin content of urine remained low (55% under the baseline value), two weeks after the fourth injection of immunotoxin. At the same time, the diuresis was increased (80% above the baseline value) and urine osmolality lowered (45% under the baseline value). When non-specific IgG replaced specific antibody, vasopressin excretion, diuresis as well as urine osmolality were unchanged. The results of this study demonstrated that the use of a specific immunotoxin results in a local interference with the vasopressinergic neurons and induces a long-term reduction of vasopressin secretion.


The use of a neurotoxic lectin, volkensin, to induce loss of identified motoneuron pools. In this study we investigated degeneration of defined motor pools in the adult rat spinal cord and the associated changes in spinal cord in dorsal root ganglia and peripheral nerve. Degeneration of motoneurons was induced by the neurotoxic lectin, volkensin. This substance is taken up by the axons and retrogradely transported to the cell body, where it inhibits proteosynthesis and kills the neuron. Accordingly, in adult Wistar rats the peroneal or the sciatic nerve was injected with 5.0 ng volkensin, and the effect of this single injection was investigated at different intervals after the operation. Retrograde labelling by horseradish peroxidase was used to reveal the extent of cell death and glial repair was studied by immunostaining with different glial cell markers. Degenerating cells were observed in the ventral horn of the lumbar spinal cord and L4 and L5 dorsal root ganglia as early as four days after volkensin treatment and by two weeks no retrogradely labelled motoneurons could be found in the treated peroneal pool. These changes were accompanied by severe muscle weight loss. Examination of the ventral horn of the spinal cord on the treated side revealed many hypertrophic astrocytes and reactive microglial cells expressing an increased level of complement receptor type 3 immunoreactivity. In the volkensin-injected peripheral nerve, distinct signs of Wallerian-like degeneration could be observed. Schwann cells identified by immunostaining to S-100 protein appeared to be preserved. Interestingly, at later stages after volkensin injection (four to eight weeks), some retrogradely labelled motoneurons were seen in the peroneal pool; their number occasionally reached 18.4% of the control pool. The dorsal root ganglia showed extensive loss of neurons and numerous abnormal neurons were found throughout the period of the study. These findings suggest that some motoneurons are able to recover from exposure to volkensin and temporary arrest of proteosynthesis. Despite this, volkensin-induced selective motoneuron death in the adult rat can be a useful experimental model for degenerative motoneuron disease.


Cis-dichlorodiammineplatinum alters GABAergic structures in the immature rat cerebellum. It has been reported that injection of the antitumoral drug cis-dichlorodiammineplatinum at 10 days of life affects cerebellar development in rats. After a single dose of 5 micrograms/g of body weight, the formation of granule cells is decreased and the maturation of postmitotic neurons is slowed down. A substantial time after treatment, reduced cell packing density of the internal granule layer and atrophy of the molecular layer can be observed. In addition, there is degeneration of some Purkinje cells and Golgi neurons. In spite of all these alterations, the regular architecture of the cerebellar folia is retained in many places. In the present study, we used immunocytochemistry with an immune serum raised against glutamic acid decarboxylase to further characterize the cis-dichlorodiammineplatinum-induced alterations of GABAergic neurons. The aim was to examine cerebellar development and to test for factors controlling the settling of GABAergic circuits. At all post-treatment intervals, most of the Purkinje and Golgi neurons and molecular layer interneurons showed stronger anti-glutamic acid decarboxylase immunoreactivity than in controls; this may have been due to altered fixation because of cis-dichlorodiammineplatinum-induced damages to the blood vessels; but could also reflect cellular retention of the enzyme, maybe due to cis-dichlorodiammineplatinum-induced damage of the microtubular apparatus. After seven days, large roundish immunoreactive varicosities were present in the molecular layer adjacent to the Purkinje cell dendritic poles. These varicosities, which were not observed in control animals, may be terminals of Purkinje cell axon recurrent collaterals contributing to the supraganglionic plexus, whose abnormal development would compensate for the reduced inhibitory inputs from inhibitory interneurons and/or Purkinje cells, which degenerated at early post-treatment intervals. At later post-treatment intervals (15 and 21 days), there were also alterations in the pericellular basket at the Purkinje cell axon hillock, which was poorly developed in or absent from the majority of cells. The finding was confirmed by morphological observation of basket cells in Golgi-Cox preparation and immunocytochemistry with an antibody raised against 200,000 mol. wt phosphorylated neurofilaments. It is concluded that early changes in anti-glutamic acid decarboxylase immunoreactivity of neurons may be due to a direct interference of the drug with the cellular metabolic pathways. The late anomalies in the anti-glutamic acid decarboxylase immunoreactivity appear to be secondary to changes in the tissue cytoarchitecture rather than being primary cis-dichlorodiammineplatinum-induced lesions of the cells.


Morphologic (CT) and functional (rCBF-SPECT) correlates in Alzheimer's disease. Thirty-five patients with a clinical diagnosis of probable Alzheimer's disease underwent computed tomography (CT) and regional cerebral blood flow (rCBF) studies using single photon emission computed tomography (SPECT). Two sets of images in each subject were scored for the extent of structure and function changes. Thirty-four of 35 patients had various degrees of atrophy on CT, 33 of whom also had perfusion deficits of varying severity. One patient with normal CT had perfusion deficits, and another patient with a normal perfusion pattern had changes on CT. Regional perfusion deficits on SPECT were seen with and without associated changes on CT. Correlations were studied between CT and SPECT scores using Spearman's rank correlation coefficients. While regional scores on CT and SPECT did not significantly correlate, the total and left hemisphere scores on two sets of images showed fair correlations (r = 0.425 and r = 0.535, respectively, P less than 0.01). The correlations between cognitive performance in patients as assessed on Mini-Mental State Examination (MMSE) and CAM-COG and perfusion scores were highly significant while CT scores showed lower correlations. These findings suggest that the relationship between structural and functional changes in Alzheimer's disease is not straightforward and that the extent of changes in function as assessed by regional cerebral blood flow studies is a reliable measure of deficits in cognitive function.


Left ventricular ejection fraction and volumes calculated from dual gated SPECT myocardial imaging with 99Tcm-MIBI. Dual gated (DG) cardiac single photon emission computed tomographic (SPECT) studies at end-diastole (ED) and end-systole (ES) were acquired in 27 ischaemic heart disease (IHD) patients after intravenous injection of 555-740 MBq 99Tcm-MIBI. Acquisition parameters were: 180 degrees from LPO to RAO, 32 projections, 64 x 64 matrix, 75 cardiac beats per projection, 80 ms at ED and 80 ms at ES for each cardiac cycle. A computer program was developed to calculate the ED and ES left ventricular (LV) volumes and LV ejection fraction (EF). The computational approach is interactive, semi-automatic and iterative with built-in visual quality control. Short axis slices are used with corresponding ED and ES slices processed as pairs from apex to base. Left ventricular cavity pixels are identified and summed on a slice-by-slice basis. Myocardial pixels are similarly identified. The computed LVEF and ED and ES volumes have been correlated with those from contrast ventriculography (CV). The mean calculated EF for 27 patients was 53.6 +/- 10.7% from DG SPECT versus 55.3 +/- 12.1% from CV (NS). The EF linear correlation coefficient was r = 0.97.


Patterns of regional cerebral blood flow in Alzheimer's disease. Regional cerebral blood flow was assessed in 35 patients with Alzheimer's disease and the same number of matched normal volunteers. Images were scored for regional perfusion deficits. Mildly demented patients had parietal and temporal perfusion deficits, often unilateral. Moderate to severely demented patients had bilateral temporal and parietal perfusion deficits. All severely demented patients also had involvement of both occipital regions. Frontal lobe deficits were seen in 14 patients. Left cortical perfusion deficits were more severe than the right cortical perfusion deficits in general. Patients with early onset of the disease showed left frontal deficits, more often, however, the total perfusion deficit scores in early and late onset groups were not statistically significant. Also, the cognitive performance scores were not statistically significant in these two groups. These results suggest a stage-dependent reduction in regional cerebral blood flow in patients with Alzheimer's disease.


Rapid quality control of 99Tcm-sestamibi. The thin layer chromatographic (TLC) method for determining the radiochemical purity of 99Tcm-sestamibi suggested by the manufacturer of the kit is slow and inconvenient. In this study a more rapid reversed phase chromatography (Sep-Pak) technique is compared with the TLC method for measurement of the radiochemical purity of 99Tcm-sestamibi. The levels of free 99Tcm-pertechnetate in 99Tcm-sestamibi were accurately determined using the Sep-Pak technique. However, due to binding of variable amounts of reduced-hydrolysed unbound 99Tcm to the chromatography cartridge, the Sep-Pak method overestimated the radiochemical purity of the radiopharmaceutical by approximately 3%. In practice, this overestimation is not important and the Sep-Pak technique can be used as a rapid method of determining radiochemical purity of 99Tcm-sestamibi.