Age-related histochemical staining patterns of glycosaminoglycans in cell nuclei of different regions of the rat brain: a pilot study

The brains of 6 rats aged 12 months (adult) and 6 rats aged 24 months (aged) were embedded in paraffin following steady state perfusion with fixation solution. Glycosaminoglycans (GAGs) were demonstrated by histochemical methods using the Alcian blue CEC method in combination with the Feulgen reaction and testis hyaluronidase. Cell nuclei revealed different patterns of GAGs in different layers of the brain cortex and in different cell types. In neuronal cell nuclei of layer 2, no GAGs are found and this may be the case also in certain types of pyramidal cells. There was a reduction of the blue staining components of the chromatin network by hyaluronidase, and also a reduction of the electronmicroscopic contrast by this enzyme in pilot study using a specimen of one animal. The enzyme effects were found to be more marked or even exclusively present in the group of aged animals. Thus, the contents of chrondroitin sulfates or hyluronate which are substrates of the enzyme may be increased either relatively or absolutely in cell nuclei of aged brains whereas GAGs resistant to the enzyme may be reduced in activity. Since GAGs are known to influence DNA activity, the variations demonstrated may be assumed to be of significance for the aging process in postmitotic cells.     

Applications of a rat multiple tissue gene expression data set

With the sequencing and assembly of the rat genome comes the difficult task of assigning functions to genes. Tissue localization of gene expression gives some information about the potential role of a gene in physiology. Various examples of the utility of multiple tissue gene expression data sets are illustrated here. First, we highlight their use in finding genes that might play an important role in a particular tissue on the basis of exclusive expression in that tissue or coexpression with a gene or genes with known function. Second, we show how this data might be used to explain known phenotypic differences between strains. Third, we show how expression patterns of genes in a genomic interval might identify candidate genes in quantitative trait loci (QTL) mapping studies. Lastly, we show how multiple tissue and species data can help researchers prioritize follow up studies to microarray experiments. All of these applications of multiple tissue gene expression data sets will play a role in functionally annotating the rat genome.     

Molecular pathology and immunology of factor VIII (hemophilia A and factor VIII inhibitors)

Factor VIII is a large procoagulant glycoprotein that circulates in plasma in a noncovalent complex with von Willebrand factor. It is essential for the efficient cleavage of coagulation factor X by factor IXa, and its absence causes a severe bleeding disorder. Plasma factor VIII is reduced from the normal range of approximately 100 to 200 ng/ml in patients with the hereditary coagulation defect, hemophilia A, as well as in patients who develop autoantibodies that inactivate factor VIII. The understanding of factor VIII structure has been enhanced by recent studies that have characterized the X chromosome gene responsible for its synthesis, and preliminary information is now available about specific genetic defects. The basis for antibody formation in approximately 15 per cent of repeatedly transfused hemophilic patients is less clear at this time, however, for these individuals appear to have a variety of genetic defects that are not characteristically different from the patients who do not develop inhibitors. Although the antibodies cause a serious problem for affected individuals, they have been very useful in characterizing normal factor VIII and nonfunctional factor VIII-like protein that is found in the plasmas of 10 per cent of patients with mild hemophilia. Moreover, they are very useful reagents that can be used for immunoassay of factor VIII that has been modified in ways that have destroyed its procoagulant function.     

Use of multiple displacement amplification to amplify genomic DNA before sequencing of the alpha and beta haemoglobin genes

AIMS: To evaluate the technique of multiple displacement amplification (MDA) for whole genome amplification from small volume blood samples before sequencing in a clinical test to identify haemoglobin gene mutations. METHODS: Phage phi29 DNA polymerase was used to perform MDA, starting with either 1 micro l of blood or 1 ng of previously isolated blood DNA from 23 patients. The amplified products were then evaluated using a clinical test that involves sequencing the haemoglobin genes to detect mutations. The results were compared with the current clinical test method that uses genomic DNA isolated using column based technology. RESULTS: The MDA technique produced large quantities (theoretically approximately 2 mg) of DNA. The amplification procedure was extremely easy and took about four hours (less than one hour of hands on technician time and three hours for amplification). When MDA products were used in the same clinical test protocol as genomic DNA isolated using column technology, there was 100% concordance for detection of a variety of point mutations in the alpha1, alpha2, and beta globin genes. CONCLUSIONS: The MDA technique is useful for overcoming the problem of insufficient genomic DNA in clinical specimens requiring haemoglobin gene sequencing and could be useful for other clinical applications.     

Prenatal diagnosis of classic hemophilia.

Prenatal diagnosis of classic hemophilia (hemophilia A) in mid-trimester was achieved by means of immunoradiometric assays for factor VIII on fetal plasma and amniotic-fluid mixtures obtained by fetoscopy. Samples were analyzed from six male fetuses at risk for severe hemophilia and from nine control fetuses for which fetoscopy was carried out to attempt prenatal diagnosis of other genetic disorders. The factor VIII coagulant-antigen values for the control (non-hemophilic) samples were 17 to 94, and the factor VIII related-antigen concentrations were 50 to 155 U per deciliter. Three of the fetuses at risk for hemophilia had factor VIII values in the control range, and these infants were normal at birth. The other three fetuses had low concentrations of factor VIII coagulant antigen but normal concentrations of factor VIII related antigen. These values and the diagnoses of severe hemophilia were confirmed with blood from the abortuses.     

Autoradiographic characterisation and localisation of 5-HT1D compared to 5-HT1B binding sites in rat brain

Summary The regional distribution and the pharmacology of the binding sites labelled with the novel 5-hydroxytryptamine (serotonin) 5-HT_1B/1D selective radioligand serotonin-O-carboxy-methyl-glycyl-[¹²⁵I]tyrosinamide (abbreviated [¹²⁵I]GTI for the sake of simplicity) was determined using quantitative autoradiography in rat brain. The distribution of [¹²⁵I]GTI binding sites was largely comparable to that of [¹²⁵I] iodocyanopindolol ([¹²⁵I] ICYP) which labels 5-HT_1B binding sites (in the presence of 8-OH-DPAT (8-hydroxy-[2N-dipropylamino]tetralin) and isoprenaline, to prevent binding to 5-HT_1A and -adrenoceptor binding sites), although a detailed analysis revealed differences.The pharmacology of the [¹²⁵I]GTI binding sites was analysed using compounds known to display high affinity for and/or distinguish between 5-HT_1B and 5-HT_1D sites: 5-carboxamidotryptamine (5-CT), sumatriptan, CP 93129 (5-hydroxy-3(4-1,2,5,6-tetrahydropyridyl)-4-azaindole), (–)pindolol, PAPP (4[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl] benzeneamine), rauwolscine, and 8-OH-DPAT. The displacement of [¹²⁵I]GTI by 5-CT was monophasic. By contrast, the selective 5-HT_1B compound CP 93129 and (–)pindolol produced biphasic curves showing a majority of high affinity sites in the globus pallidus and the substantia nigra, whereas PAPP and sumatriptan (which are somewhat 5-HT_1D selective) produced biphasic curves indicating a minority of high affinity sites in these areas. In addition, by blocking the 5-HT_1B sites with 100 nM CP 93129, the remaining population of [¹²⁵I]GTI binding sites could be studied and was found to have high affinity for PAPP, rauwolscine and 8-OH-DPAT. The pharmacological profile of the major binding component was typical of the 5-HT_1B type: 5-CT > CP 93129 (–)pindolol > sumatriptan >/ PAPP > rauwolscine. The profile of the minor component of [¹²⁵I] GTI binding is best characterised as that of a 5-HTID site: 5-CT > PAPP sumatriptan > rauwolscine > (–)pindolol CP 93129.The localisation of the non 5-HT_1B [¹²⁵I]GTI binding sites was characterised by blocking the 5-HT_1B receptors with 100 nM CP 93129. Low densities of the 5-HT_1D recognition sites were found to be present in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra (reticular part), nuclei of the (normal and accessory) optic tract, different nuclei of the geniculate body and frontoparietal cortex, although higher densities of 5-HT_1B sites were always observed in the same structures. Thus, in agreement with the recent cloning of a rat 5-HT_1D receptor cDNA, the presence and the distribution of 5-HT_1D sites could be documented in rat brain. However, when compared to 5-HT_1B sites, 5-HT_1D sites represent only a minor component of the [¹²⁵I]GTI binding in the rat brain structures studied.Correspondence to: D. Hoyer at the above address     

Brain somatostatin: a candidate inhibitory role in seizures and epileptogenesis

Recent evidence shows that neuropeptide expression in the CNS is markedly affected by seizure activity, particularly in the limbic system. Changes in neuropeptides in specific neuronal populations depend on the type and intensity of seizures and on their chronic sequelae (i.e. neurodegeneration and spontaneous convulsions). This paper reviews the effects of seizures on somatostatin-containing neurons, somatostatin mRNA and immunoreactivity, the release of this peptide and its receptor subtypes in the CNS. Differences between kindling and status epilepticus in rats are emphasized and discussed in the light of an inhibitory role of somatostatin on hippocampal excitability. Pharmacological studies show that somatostatin affects electrophysiological properties of neurons, modulates classical neurotransmission and has anticonvulsant properties in experimental models of seizures. This peptidergic system may be an interesting target for pharmacological attempts to control pathological hyperactivity in neurons, thus providing new directions for the development of novel anticonvulsant treatments.     

Characterisation of the fish sst3 receptor, a member of the SRIF1 receptor family: atypical pharmacological features

The first cloned non-mammalian somatostatin (somatostatin release-inhibiting factor = SRIF) receptor previously obtained from the teleost fish Apteronotus albifrons and generically named somatostatin receptor 3 (fsst3), was stably expressed and characterised in Chinese hamster lung fibroblast (CCL39) cells. Radioligand binding studies were performed with four radioligands selective for SRIF receptors in CCL39 cells expressing the fsst3 receptors; [125I]LTT-SRIF28 ([Leu8, D-Trp22, 125I-Tyr25]-SRIF28), [125I]Tyr10-cortistatin, [125I]CGP 23996, and [125I]Tyr3-octreotide labelled the fsst3 receptor with high affinity (pKd values: 10.47, 10.87, 9.59 and 9.57) and in a saturable manner, but defined different Bmax values; 4500, 4000, 3400 and 1500 fmol/mg, respectively. The affinities of SRIF peptides and analogues determined for fsst3 receptors displayed the following rank order of potency: seglitide = SRIF25 > SRIF14 = SRIF28 > cortistatin 14 > BIM 23014 > RC160 = L361,301 = octreotide > or = BIM 23052 > or = L362,855 > CGP23996 > BIM 23056 > BIM 23030 = cycloantagonist > SRIF22. The pharmacological profiles determined with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I]Tyr10-cortistatin correlated highly significantly (r = 0.96-0.99), whereas [125I]Tyr3-octreotide binding was rather divergent (r = 0.78-0.81). Further, [125I]Tyr3-octreotide- and [125I]CGP 23996-labelled sites showed higher affinity for the various peptides than [125I]LTT-SRIF28 and [125I]Tyr10-cortistatin-labelled sites, although there were exceptions. [125I]LTT-SRIF28-binding to fsst3 receptors and human sst1-5 receptors was compared; the fsst3 binding profile correlated better with the hsst5- than with the hsst3 receptor profile. SRIF inhibited potently forskolin-stimulated adenylate cyclase activity in fsst3 transfected CCL39 cells; this effect was blocked by pertussis toxin, suggesting coupling of the fsst3 receptor to Gialpha and/or Goalpha. [125I]LTT-SRIF28 binding was detected in fish brain, liver, heart, spleen, and stomach, but not in gut. The pharmacological profile of [125I]LTT-SRIF28-labelled sites in brain, but not in liver, correlated significantly with the recombinant fsst3 receptor, in agreement with expression of the fsst3 receptor gene found by RT-PCR in the brain. However, biphasic binding curves obtained with two SRIF-analogues in brain, as well as the distinct pharmacological profile of the liver SRIF receptor, suggest the existence of several yet to be defined SRIF receptor subtypes in fish. The present data demonstrate that the recombinantly expressed fsst3 receptor has a pharmacological profile compatible with that of a SRIF1 receptor, although the rank order of affinity of fsst3 is closer to that of hsst5 than hsst3 receptors, as may be found when comparing very distantly related species. The fsst3 receptor expressed in CCL39 cells, is negatively coupled to adenylate cyclase activity via pertussis toxin-sensitive G-proteins, like mammalian sst3 receptors. Radioligand binding performed with fish tissue suggests the presence of a native sst3 receptor in brain as well as other yet to be defined SRIF receptor subtypes.     

Characterisation of human recombinant somatostatin receptors. 4. Modulation of phospholipase C activity

Total [3H]phosphoinositide (IPx) accumulation, a measure of phospholipase C (PLC) activity, induced by somatostatin (somatotropin release-inhibiting factor, SRIF) and cortistatin (CST) analogues was studied at human somatostatin receptor subtypes 1-5 (hsst1-5) recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. SRIF14 (10 microM) stimulated total [3H]-IPx production 200% and 1070% over basal levels, and increased intracellular Ca2+ ([Ca2+]i) 1600% and 2790%, in cells expressing hsst3 and hsst5 receptors, respectively. The SRIF14-stimulated IPx production was partly blocked by 100 ng/ml pertussis toxin (PTX) (30% and 15% inhibition, respectively). At hsst1, hsst2, and hsst4 receptors, only weak or no stimulation of PLC activity was found (Emax = 114%, 122%, and 102%, respectively). Consequently, hsst3 and hsst5 receptors were subjected to more detailed studies to establish pharmacological profiles of PLC stimulation. At hsst3 receptors, the relative efficacies of most ligands were in the same range (maximum response Emax = 218-267%). At hsst5 receptors Emax varied over a broad range, seglitide, CST17, SRIF28 displaying almost full agonism compared to SRIF14, whereas octreotide and BIM 23052 showed very low partial agonism. BIM 23056 behaved as an antagonist on SRIF14-induced total [3H]-IPx accumulation with a pKB (negative logarithm of antagonist binding constant) of 6.74 at hsst3 receptors, and of 6.94 at hsst5 receptors. The putative cycloantagonist SA showed weak antagonist activity on SRIF14-induced total [3H]-IPx levels at hsst3 (pKB = 5.85), but not at hsst5 receptors. The [3H]-IPx accumulation profiles at sst3/sst5 receptors were compared to their respective radioligand binding ([125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide binding), to [35S]GTPgammaS binding, and to forskolin-stimulated adenylate cyclase (FSAC) inhibition profiles determined previously in CCL39 cells. The different affinity profiles correlated relatively well at both receptor subtypes with PLC activation (sst3: r = 0.90-0.97; sst5: r = 0.80-0.87). However, [35S]GTPgammaS binding correlated only minimally with stimulation of [3H]-IPx levels at sst5 receptors (r = 0.59), but rather well at sst3 receptors (r = 0.80). A moderate correlation was also observed between inhibition of FSAC activity and stimulation of PLC activity for hsst3 and hsst5 receptors with correlation coefficients of 0.85 and 0.70, respectively. In summary, most SRIF analogues behave as full agonists at hsst3 receptors and agonist-induced phosphoinositide turnover correlates well with radioligand binding, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, all measured in CCL39 cells. By contrast, at hsst5 receptors, most SRIF analogues behave as intermediate or very low partial agonists (although receptor levels are comparatively high, 7000 vs. 400 fmol/mg), and the agonist-induced phosphoinositide turnover correlates rather poorly with radioligand binding, [35S]GTPgammaS binding or inhibition of adenylate cyclase activity, all measured in the same cell line. Agonist-induced phosphoinositide turnover, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, show differences both in the rank orders of potency and relative efficacy at hsst3 and markedly at hsst5 receptors, suggesting either that PLC activity is functionally irrelevant or, more probably, that agonist-dependent receptor trafficking is taking place in CCL39 cells.