Molecular diagnosis of transthyretin Met30 mutation in an Italian family with familial amyloidotic polyneuropathy.

We report the molecular analysis of the transthyretin gene in a large Italian pedigree with familial amyloidotic polyneuropathy and demonstrate the presence of a Met30 mutation. The usefulness of the genetic analysis in the identification of presymptomatic persons and the diagnosis of individuals with partial symptoms is discussed.

Different localization of spermidine/spermine N1-acetyltransferase and ornithine decarboxylase transcripts in the rat kidney.

In situ hybridization histochemistry of transverse sections from male rat kidney showed that the mRNA of the regulatory enzyme of polyamine degradation, spermidine/spermine N1-acetyltransferase, has a spotty distribution in the cortex, is low and diffused in the outer stripe and high and diffused in the inner stripe of the outer medulla. At the cellular level, this mRNA is solely expressed by the epithelium of the distal straight and convoluted nephron tubules. Since biosynthetic ornithine decarboxylase mRNA is solely found in the proximal straight tubules, it is proposed that polyamine biosynthesis and degradation occur at separate sites along the nephron.

Gene relaxation and aging: changes in the abundance of rat ventral prostate SGP-2 (clusterin) and ornithine decarboxylase mRNAs.

Sulfated glycoprotein 2 (SGP-2) mRNA progressively increased in the ventral prostate of the aging rat, reaching, at 24 months, 4-fold higher than at 3 months. Ornithine decarboxylase (ODC) mRNA peaked at 6 months (4-fold increase), and at 12 and 24 months was maintained at higher levels than at 3 months. ODC enzymatic activity was enhanced at 6 months to a much smaller extent than its own mRNA, the values at 12 and 24 months dropping to below those at 3 months. Putrescine (Put), spermidine (Spd) and spermine (Sp) concentrations also peaked at 6 months (100% increase for Put, 50% for Sp and Spd). At 24 months, Put and Spd were diminished, and Sp was unchanged with respect to the 3-month values. Under the same conditions, glyceraldehyde-3-phosphate dehydrogenase mRNA did not undergo significant alterations.

Microanatomical changes of intracerebral arteries in spontaneously hypertensive rats: a model of cerebrovascular disease of the elderly.

Changes occurring in intracerebral arteries of 24-week-old spontaneously hypertensive rats (SHR) compared with age-matched normotensive Wistar-Kyoto (WKY) rats were assessed using microanatomical techniques associated with image analysis. Morphometric parameters investigated included arterial diameter, lumen area, wall area, and wall-to-lumen ratio. Intracerebral arteries (lumen diameter>46 microm) and arterioles (lumen diameter 46-10 microm) of frontal cortex, striatum, and hippocampus were examined. In frontal cortex of SHR arterial wall hypertrophy and luminal narrowing were observed. In striatum, an increase of wall area not accompanied by luminal narrowing predominates resulting in arterial hypertrophy without vasoconstriction. In hippocampal arteries of SHR, luminal narrowing, without changes of wall area was found indicating the occurrence of remodeling. In brain areas investigated, hypertensive changes affected primarily arterioles. The demonstration of a sensitivity of intracerebral arteries to hypertension suggests that changes of these vessels may represent a cause of brain structural alterations occurring in hypertension. The specificity of alterations occurring in intracerebral arteries of brain areas investigated may account for the different localization of cerebral lesions in cerebrovascular disease. The possibility that microanatomical changes developed in intracerebral arteries of SHR may represent a model of cerebrovascular disease of the elderly is discussed.

Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.

BACKGROUND: Progressive supranuclear palsy (PSP) is characterized by a pure neurofibrillary tau pathology involving mainly basal ganglia and brainstem nuclei. In addition to a haplotype of the tau gene potentially favoring tau aggregation, lipoperoxidation has been shown to be associated with PSP tau pathology. OBJECTIVE: To analyze cdk5/p35 complex, a kinase that regulates neurite outgrowth, as a potential cellular mechanism underlying tau phosphorylation in brain tissues from PSP and control cases and comparatively in cerebral cortex from subjects with AD. METHODS: Cdk5/p35 protein levels and distribution were evaluated by immunoblotting and immunocytochemistry in brain regions from seven PSP, six AD, and seven control cases, with similar postmortem intervals. RESULTS: Total cdk5 protein levels were significantly increased by more than threefold in PSP tissue and were augmented in PSP neurons, codistributed with tau immunoreactivity. P35, the regulatory subunit of cdk5, was degraded by postmortem proteolysis to the same extent in PSP, AD, and control tissues. CONCLUSIONS: The proteolysis in vivo of p35, the regulatory subunit of the kinase, is not ascertainable because it is masked by its postmortem degradation. The study, however, indicates that in PSP, the alteration of cdk5 is different from that described in AD and suggests that the absence of amyloid beta protein deposition may account for the different pathways responsible for the same kinase activation.

Immunological properties of ribosome-inactivating proteins and a saporin immunotoxin.

Antibodies have been raised in rabbits against plant ribosome-inactivating proteins (RIPs) and used to demonstrate cross-reactivity between RIPs from plants belonging to the same family, but little or no cross-reactivity between RIPs from taxonomically unrelated plants. When an immunotoxin consisting of saporin conjugated to bovine IgG was injected into rabbits, the animals formed antibodies against both saporin and bovine IgG.

Changes of retinal neurons and glial fibrillary acid protein immunoreactive astrocytes in spontaneously hypertensive rats.

OBJECTIVE: The influence of arterial hypertension on retinal neurons and glial fibrillary acid protein (GFAP) immunoreactive astrocytes was investigated in spontaneously hypertensive rats (SHRs). METHODS: The retinas of 4- and 6-month-old SHRs and age-matched Wistar-Kyoto rats (WKY) were investigated. A group of SHRs, treated from 4 to 6 months with the hypotensive drug hydralazine, was also examined. Microanatomical and immunohistochemical techniques associated with image analysis and the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) technique for apoptosis or necrosis were used, as well as astrocyte molecular biology (Western blot) techniques. RESULTS: In 4-month-old SHR and WKY rats, retinal morphology and the number of retinal neurons and of GFAP-immunoreactive astrocytes were similar, with the exception of the occurrence of 1% of TUNEL-positive ganglionic neurons in SHRs. In 6-month-old SHRs a decrease of retinal volume and of the number of ganglionic neurons and photoreceptors was observed, compared with age-matched normotensive WKY rats or younger SHR and WKY rats. Two per cent of ganglionic neurons and 5% of photoreceptors were also TUNEL positive. In 6-month-old SHRs, hypertrophic perivascular GFAP-immunoreactive astrocytes were found, whereas their number was unchanged compared to younger cohorts or WKY rats. An increased expression of GFAP was also noticeable in SHRs by Western blot analysis. Hypotensive treatment with hydralazine partly countered retinal changes occurring in SHRs. CONCLUSIONS: The occurrence of neuronal and astroglial changes when a stable hypertension was developed, and their sensitivity to antihypertensive treatment, suggest that they may represent a hypertension-related phenomenon.

Nonsteroidal antiinflammatory agents. 2. Synthesis and biological activity of 2-chloroindolecarboxylic acids.

The Vilsmeier and the Arndt-Eistert reactions have been employed for the synthesis of 1-phenyl-2-chloroindole-3-acetic acid (4). The antiinflammatory activity of 2-chloroindole-3-carboxylic acid (1), 1- methyl-2-chloroindole-3-carboxylic acid (2), 1-phenyl-2-chloroindole-3-carboxylic acid (3), and 4 was compared with the activity of indomethacin in the carrageenin rat edema. The best results are given by compounds 1 and 2 bearing H or CH3 at position 1 and COOH at position 3.

The hippocampus in spontaneously hypertensive rats: a quantitative microanatomical study.

The influence of hypertension on the morphology of hippocampus was assessed in spontaneously hypertensive rats of two, four and six months and in age-matched normotensive Wistar-Kyoto rats. Values of systolic pressure were slightly increased in two-month-old spontaneously hypertensive rats in comparison with age-matched Wistar-Kyoto rats and augmented progressively with age in spontaneously hypertensive rats. No microanatomical changes were observed in the hippocampus of spontaneously hypertensive rats of two months in comparison with age-matched Wistar-Kyoto rats, whereas a decrease of white matter volume was observed in the CA(1) subfield and in the dentate gyrus of four-month-old spontaneously hypertensive rats. In the hippocampus of six-month-old spontaneously hypertensive rats a reduction of grey matter volume both in the CA(1) subfield and in the dentate gyrus, a loss of neurons affecting to a greater extent the CA(1) subfield and an increase of glial fibrillary acid protein-immunoreactive astrocytes was found. The occurrence of apoptosis and/or necrosis identified using the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick end labelling technique was also observed in the CA(1) subfield and to a lesser extent in the dentate gyrus. The only change noticeable in the CA(3) subfield of six-month-old spontaneously hypertensive rats was a slight increase in the number of glial fibrillary acid protein-immunoreactive astrocytes. These findings indicate the occurrence of neuronal loss and of astrocyte changes in the hippocampus of spontaneously hypertensive rats of six months, being the CA(1) subfield the area most affected. The relevance of these neurodegenerative changes in hypertension and the possible occurrence of apoptosis and/or necrosis as expression of hypertensive brain damage is discussed.

In vitro studies of the biosynthesis of brain tubulin on membranes.

Membrane elements in brain tissue contain relatively large amounts of alpha- and beta-tubulin (FIGURES 2 and 3). We have investigated the subcellular sites of tubulin biosynthesis in order to determine the origin of this membrane-associated tubulin. Free and membrane-bound polysomes from rat forebrain were separated by differential centrifugation, and the products of translation from these polysome populations were analyzed by 2DGE (FIGURES 4 and 6). Alpha- and beta-tubulin subunits were synthesized by the free polysome population (FIGURES 4 and 5A and B). The membrane-bound polysome fraction synthesized a protein with similar (but not identical) characteristics to alpha-tubulin (denoted as "MB" in FIGURE 6), including isoelectric point, molecular weight, peptide map, and copurification with microtubules after aggregation-disaggregation. Tubulin subunits synthesized in vitro by free polysomes could associate posttranslationally with a microsome fraction (FIGURE 7A). The association of the tubulin translation products with membranes was not disrupted by high salt; the associated tubulin, however, was susceptible to proteolytic digestion, with the exception of one of the beta-tubulin subunits (FIGURE 7B). There was an identical protease-resistant beta-tubulin subunit among the native proteins of the smooth microsome fractions. Our data is consistent with the conclusion that at least one beta subunit of membrane-associated tubulin is synthesized by free polysomes and becomes posttranslationally added to membrane structures. It is unlikely that a cotranslational mechanism is responsible, in which there is a signal-mediated insertion of a growing polypeptide chain to membrane. Our results, however, are consistent with a "membrane trigger" mechanism proposed by Wickner in which the membrane lipid bilayer triggers the folding of a polypeptide into a configuration that allows integral membrane insertion. The association of tubulin with membranes may also be secondary to the interaction of hydrophobic elements. The amino acid sequence of beta tubulin is known to contain several hydrophobic domains. Tubulin can be incorporated into phospholipid vesicles and various subcellular membrane elements. In our studies, in vitro synthesized tubulin from free polysome was found to be purified by hydrophobic affinity chromatography with ethane-sepharose (FIGURE 8). Thus, the hydrophobic characteristics of newly synthesized tubulin could be partially responsible for the posttranslational association of tubulin subunit with membranes. Native tubulin in a soluble fraction of CNS tissue was not purified by hydrophobic affinity chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties and function of brain carbonic anhydrase.

This chapter has described the characterization and biogenesis of soluble and membrane-bound CA in the central nervous system. The two forms of the enzyme appear to be quite similar in their molecular characteristics, however the data strongly indicate that they are synthesized on separate polysomal populations; the membrane-bound form resulting from synthesis on the RER. Our preliminary data suggest that the partitioning of mRNA for CA on the different polysomes results from the interaction of partial nascent chains with a specific receptor on the RER. We feel a function of membrane-associated synthesis is for the targeting of CA to sites in the cell where there are enzymes that can rapidly utilize the protons and bicarbonate produced by CA catalytic activity for ion exchange reactions. We have also presented arguments that CA may function as a bicarbonate source in the control of metabolism specifically in the acceleration of fatty acid synthesis in the oligodendrocyte.

Neuronal loss up-regulates clusterin mRNA in living neurons and glial cells in the rat brain.

The aim of this study was to examine the time course and cellular localization of clusterin mRNA after neurodegeneration. Selective neuronal death was achieved in the rat inferior olivary complex after volkensin injection in the contralateral cerebellar cortex. Quantitative analysis of the in situ hybridization signal demonstrated over-expression of clusterin mRNA in living neurons at 6 days and outside the neuronal cell bodies at 10 days post-injection. We conclude that, in our experimental model, clusterin over-expression occurs as an early and transient neuronal and as a delayed glial response to selective neuronal death, supporting the view that clusterin may be involved in cytoprotection and tissue remodeling.

Suicide retrograde transport of volkensin in cerebellar afferents: direct evidence, neuronal lesions and comparison with ricin.

Volkensin and ricin, either free or conjugated with colloidal gold, were injected into the cerebellar cortex of rats. The inferior olive and pontine nuclei were examined to verify the retrograde axonal transport of these two toxins, and the consequent neuronal damage. No evidence was obtained of a retrograde axonal transport of ricin in these pathways. Injection of gold-conjugated volkensin in the cerebellar cortex resulted in retrogradely labelled neurones in the inferior olive after 3 h, and in the pontine nuclei after 6 h. Degenerative changes were very severe in the retrogradely labelled neurones 48 h after the gold-conjugated volkensin injection. In the Nissl-stained material, neuronal degeneration started to be evident in the inferior olive 12 h, and in pontine nuclei 6 h, after volkensin injection. The neuronal degeneration in both the inferior olive and pons increased up to 4 days after the injection. These findings provide direct evidence of the retrograde axonal transport of volkensin in the central nervous system, and the time course of the consequent degenerative changes in the afferents to the cerebellar cortex.

Sununit structure of synaptosomal tubulin.

The subunit structure of rat brain synaptosomal tubulin was examined by high resolution two-dimensional gel fractionation. Whole brain cytoplasmic tubulin consists of two groups of alpha subunits (alpha1 and alpha2), and a minimum of two beta subunits (beta1 and beta2). Both alpha subunits consist of a major relatively acidic form and minor relatively basic forms. In contrast, tubulin purified from synaptoplasm contains an additional subunit, alpha3, which has the same isoelectric point but slightly faster electrophoretic mobility than alpha1 and alpha2. All synaptosomal alpha subunits are the relatively acidic forms and the minor basic forms are absent. The synaptosomal beta subunits have electrophoretic properties similar to the corresponding cytoplasmic forms. The alpha3 synaptosomal tubulin subunit has affinity for colchicine, has a tryptic peptide map similar to whole brain cytoplasmic alpha tubulin, and can be purified by a standard tubulin purification method.

The subunit composition of cerebellar tubulin: evidence for multiple beta tubulin messenger RNAs.

Cytoplasmic proteins were isolated from adult rat forebrain and cerebellum and analyzed by two-dimensional gel electrophoresis under conditions which the major subunits of tubulin were separated. Forebrain cytoplasmic tubulin consisted of two groups of alpha subunits (alpha 1 and alpha 2) and a minimum of two beta subunits (beta 1 and beta 2). However, the rat cerebellar cytoplasmic proteins contained greatly decreased amounts of the beta 1 tubulin subunit relative to the analysis of forebrain proteins. Messenger RNA (mRNA) was purified from cerebellum and forebrain and translated in wheat germ homogenate. Analysis of the translation products of cerebellar mRNA indicated only a trace amount of the beta 1 subunit, whereas the expected amount of beta 1 was found among the translation products of forebrain mRNA. This data is consistent with the conclusion that the beta 1 and beta 2 subunits of tubulin are synthesized from different mRNAs. A decrease in beta 1 mRNA relative to other tubulin mRNAs may be one of the factors responsible for the low steady state amounts of beta 1 tubulin in the cerebellum.

The inhibitory effect of methergoline on LAE-32-induced head-twitches in mice.

Methergoline (a selective 5-HT blocker) was more powerful than chlorpromazine in inhibiting LAE-32-induced head-twitches in mice. Suggestions are made regarding the possible antihallucinogenic effect of this drug.

Effect of methergoline on body temperature in mice.

Serotonin (5-HT) involvement in body temperature regulation has been studied in mice by means of a 5-HT-selective blocking agent (methergoline). This drug causes an effect on body temperature which is dependent on environmental temperature. At environmental temperatures of 25 degrees C and 11 degrees C methergoline has a hypothermic effect, while at 36 degrees C environmental temperature, methergoline has a hyperthermic effect. At 25 degrees C environmental temperature, the hypothermic effect induced by 125 mug/kg i.p. of methergoline could be antagonized by low doses of LAE-32 (80 mug/kg s.c.), while there was not such an antagonism using higher doses of LAE-32 (100 and 300 mug/kg s.c.). This has been explained using Jalfre's hypothesis of the existence of 5-HT inhibitory and excitatory receptors.

Glial reaction to volkensin-induced selective degeneration of central neurons.

Volkensin, a highly toxic protein retrogradely transported through axons, was used to target primary neuronal death in brainstem precerebellar relays after injection in the cerebellar cortex of rats. The reaction of astrocytes and microglia was studied with immunohistochemistry in the inferior olivary and pontine nuclei from 6 h to 14 days. Neurodegenerative features were evident since the first hours, especially in the pontine nuclei, and neuronal loss reached a plateau at 7 days in the inferior olive and at 10 days in the pons. Astrocytic activation, revealed by glial fibrillary acidic protein immunoreactivity, was concomitant with early signs of neuronal death and gradually increased. Microglia activation, revealed by OX-42 immunoreactivity, was evident at 2 days and became rapidly intense in precerebellar relays. At 1 week, marked ED-1 immunoreactivity also revealed phagocytic features of microglia, which persisted during the second week. In addition, major histocompatibility complex antigens (MHC) class I and II were induced in cells exhibiting microglial features. In the inferior olive, MHC I immunoreactivity was evident since 4 days and persisted at 14 days, whereas MHC II induction was intense at 7 days and subsided at 2 weeks. In the pontine nuclei high expression of both MHC antigens persisted instead at 14 days, probably reflecting the progression of neuronal death. Thus, targeted lethal injury of central neurons elicited prompt activation of both astrocytes and microglia; the marked microglia activation resulted in phagocytic features and immunophenotypic changes, with a temporal regulation that paralleled the evolution of neurodegenerative phenomena.

Muscarinic cholinergic receptors in the human right coronary artery: a receptor binding and autoradiographic study.

We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [3H]-quinuclidinyl benzilate (QNB) binding sites in the human right coronary artery. The ligand was bound to sections of the human right coronary artery in a manner consistent with the labelling of muscarinic receptors. The addition of pirenzepine or of carbachol to the incubation medium to generate displacement curves was indicative of the presence of M1 and M2 receptors in the right coronary artery. Autoradiography showed the localization of M1 sites primarily in the medial layer of the right coronary artery. M2 sites were located primarily in the adventitia. No [3H]-QNB binding sites were observed in the endothelium. A possible role of muscarinic receptors in the pathogenesis of coronary vasospasm is discussed.

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.