Differential expression of laminin and fibronectin and of their related metalloproteinases in human glioma cell lines: relation to invasion.

In the present work, we analyzed the expression of two major components of the extracellular matrix (ECM), laminin and fibronectin and of two related matrix-metalloproteinases, MMP-2 and MMP-9, in three human glioma cell lines (8 MG, 42 Mg and GL-15) in relation with their differential invasive properties. Immunocytochemistry and Western-blots assays indicated the presence of a 200 kDa laminin, similarly expressed in the three cell lines but undetectable in their ECM. In the opposite, a 230 kDa fibronectin, detected in the three cell lines was differently expressed and only observed in the ECM of the less invasive 8 and 42 MG cells. MMP-2 mRNA analyzed by Northern blots and proMMP-2, evaluated by zymography, were found in the three cell lines but were both ten times higher in the most invasive GL-15 cells. In addition, the active form of MMP-2 was only found in the GL-15 cells. In the opposite, the expression of specific tissular inhibitor (TIMP)-2, an endogenous MMP-2 inhibitor, was restricted to the less invasive cells. MMP-9 activity was detected only in the 8 and 42 MG cells and may not be directly involved in invasion. Taken together, these results indicate that a high MMP-2/TIMP-2 ratio may be responsible for the absence of extracellular fibronectin, underlining the participation of tumour cells in the proteolytic degradation of the ECM. An unbalanced MMP-2/TIMP-2 ratio in the micro-environment of malignant cells may contribute to their invasive properties.

Ciliary neurotrophic factor may activate mature astrocytes via binding with the leukemia inhibitory factor receptor.

Ciliary neurotrophic factor (CNTF) acts on immature astrocytes that express its trimeric receptor. In contrast, mature astrocytes do not significantly express the specific CNTFalpha receptor subunit, yet they respond to CNTF administration in vivo. Here we show that this controversy may be solved by a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine on mature astrocytes. A convergent set of results supports the hypothesis that the CNTF effect is due to the illegitimate binding on the leukemia inhibitory factor receptor (LIFR): (i) it requires high concentration of recombinant rat CNTF; (ii) it involves the Jak/Stat and Ras-MAPK pathways; (iii) it is preserved in CNTFRalpha-/- cells; (iv) it is potentiated by soluble CNTFRalpha added to the medium; and (v) it is significantly decreased by a partial antagonist of LIFR. On these bases, we propose a mechanistic model in which, in the adult brain, a CNTF/LIFR interglial system may be modulated by neurons that synthesize CNTFRalpha.

Metabolism of methoxymorpholino-doxorubicin in rat, dog and monkey liver microsomes: comparison with human microsomes.

The morpholino anthracycline, methoxymorpholino-doxorubicin (MMDx) is a novel anticancer agent. The metabolism of this highly lipophilic doxorubicin analogue is not fully elucidated. MMDx is metabolically activated in vivo, resulting in an 80-fold increase in potency over the parent drug. In this study, MMDx in vitro metabolism was compared in rat, dog, monkey and human liver microsomes. When microsomal fractions were incubated with MMDx, 6-8 metabolites were formed depending on the species and on the substrate concentrations. Among these eight metabolites, three comigrated with authentic standards, namely MMDx-ol, PNU156686 and PNU159682, and the five others are in the process of being characterized. Quantitatively, monkey and human metabolize MMDx with a higher rate than rat and dog. Qualitatively, MMDx metabolic profile in dog microsomes was different from the three other species. MMDx-ol was predominant in dog and only minor in other species. In conclusion, MMDx metabolism was species-different. Rat and monkey liver microsomes may be used as models to study MMDx metabolism in humans. Dog liver microsomes may be a good model for studying the formation of MMDx-ol.

Inorganic polyphosphate: a molecule of many functions.

Inorganic polyphosphate (poly P) is a chain of tens or many hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite inorganic polyphosphate's ubiquity--found in every cell in nature and likely conserved from prebiotic times--this polymer has been given scant attention. Among the reasons for this neglect of poly P have been the lack of sensitive, definitive, and facile analytical methods to assess its concentration in biological sources and the consequent lack of demonstrably important physiological functions. This review focuses on recent advances made possible by the introduction of novel, enzymatically based assays. The isolation and ready availability of Escherichia coli polyphosphate kinase (PPK) that can convert poly P and ADP to ATP and of a yeast exopolyphosphatase that can hydrolyze poly P to Pi, provide highly specific, sensitive, and facile assays adaptable to a high-throughput format. Beyond the reagents afforded by the use of these enzymes, their genes, when identified, mutated, and overexpressed, have offered insights into the physiological functions of poly P. Most notably, studies in E. coli reveal large accumulations of poly P in cellular responses to deficiencies in an amino acid, Pi, or nitrogen or to the stresses of a nutrient downshift or high salt. The ppk mutant, lacking PPK and thus severely deficient in poly P, also fails to express RpoS (a sigma factor for RNA polymerase), the regulatory protein that governs > or = 50 genes responsible for stationary-phase adaptations to resist starvation, heat and oxidant stresses, UV irradiation, etc. Most dramatically, ppk mutants die after only a few days in stationary phase. The high degree of homology of the PPK sequence in many bacteria, including some of the major pathogenic species (e.g. Mycobacterium tuberculosis, Neisseria meningitidis, Helicobacter pylori, Vibrio cholerae, Salmonella typhimurium, Shigella flexneri, Pseudomonas aeruginosa, Bordetella pertussis, and Yersinia pestis), has prompted the knockout of their ppk gene to determine the dependence of virulence on poly P and the potential of PPK as a target for antimicrobial drugs. In yeast and mammalian cells, exo- and endopolyphosphatases have been identified and isolated, but little is known about the synthesis of poly P or its physiologic functions. Whether microbe or human, all species depend on adaptations in the stationary phase, which is truly a dynamic phase of life. Most research is focused on the early and reproductive phases of organisms, which are rather brief intervals of rapid growth. More attention needs to be given to the extensive period of maturity. Survival of microbial species depends on being able to manage in the stationary phase. In view of the universality and complexity of basic biochemical mechanisms, it would be surprising if some of the variety of poly P functions observed in microorganisms did not apply to aspects of human growth and development, to aging, and to the aberrations of disease. Of theoretical interest regarding poly P is its antiquity in prebiotic evolution, which along with its high energy and phosphate content, make it a plausible precursor to RNA, DNA, and proteins. Practical interest in poly P includes many industrial applications, among which is the microbial removal of Pi in aquatic environments.

Fetal striatal allografts reverse cognitive deficits in a primate model of Huntington disease.

Substitutive therapy using fetal striatal grafts in animal models of Huntington disease (HD) have already demonstrated obvious beneficial effects on motor indices. Using a new phenotypic model of HD recently designed in primates, we demonstrate here complete and persistent recovery in a frontal-type cognitive task two to five months after intrastriatal allografting. The striatal allografts also reduce the occurrence of dystonia, a major abnormal movement associated with HD. These results show the capacity of fetal neurons to provide a renewed substrate for both cognitive and motor systems in the lesioned adult brain. They also support the use of neural transplantation as a potential therapy for HD.

Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli.

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB.

Brainstem neurons projecting to the rostral ventral respiratory group (VRG) in the medulla oblongata of the rat revealed by co-application of NMDA and biocytin.

Groups of neurons in the medulla and pons are essential for the rhythm generation, pattern formation and modulation of respiration. The rostral Ventral Respiratory Group (rVRG) is thought to be a crucial area for rhythm generation. Here we co-applied biocytin and NMDA in the rVRG to label retrogradely brainstem neurons reciprocally connected to a population of inspiratory neurons in the rat rVRG. The procedure excited rVRG neurons in multi-unit recordings and led to a Golgi-like labelling of distant cells presumably excited by efferents from the rVRG. Injection of biocytin without NMDA did not label neurons in distant structures. Several brainstem ipsi- and contralateral structures were found to project to the rVRG, but three major respiratory-related structures, the nucleus of the solitary tract (NTS), the parabrachialis medialis and Kölliker-Fuse nuclei (PB/KF) and the caudal VRG, which are known to project bilaterally to the rVRG, were exclusively labelled ipsilaterally, suggesting an ipsilateral excitation of these structures by the rVRG. The pathways of efferent axons from labelled neurons in the rVRG were traced rostrally towards the pons and caudally to the spinal cord. Terminal axonal arborizations were seen in the same regions where retrogradely filled neurons were found as well as in a few other motor nuclei (the dorsal vagal motor nucleus and XII nucleus). Moreover, in the NTS and the PB/KF, efferent terminal varicosities were seen closely apposed to the soma and proximal dendrites of labelled neurons, suggesting monosynaptic connections between the rVRG and these nuclei.

An in vitro microassay to assess the ability of Plasmodium falciparum-infected erythrocytes to bind to the human syncytiotrophoblast.

PROBLEM: Erythrocytes parasitized by matures stages of Plasmodium falciparum are frequently sequestered in human placenta. The consequences of this sequestration have been well described, but little is known about the mechanisms used by the parasite to concentrate in the placenta. METHOD OF STUDY: We developed an in vitro assay to study their binding capacity to the human syncytiotrophoblast. Our cytoadherence test was scaled down, and each step of the assay was optimized to enhance the sensitivity of our model. RESULTS: Cytoadherence assays between P. falciparum-infected erythrocytes and human trophoblasts are easily performed using low numbers of trophoblast cells. The process can also be used to carry out immunofluorescence and immunostaining techniques. CONCLUSIONS: The test may be adapted to any kind of cell, is inexpensive, and allows the culture of virtually any kind of cell for several weeks.

Isotypic analysis of maternally transmitted Plasmodium falciparum-specific antibodies in Cameroon, and relationship with risk of P. falciparum infection.

In malaria-endemic areas, infants are relatively protected against malaria infection. Such protection is though to be related principally to the transplacental transfer of maternal antibodies. We measured total and Plasmodium falciparum-specific IgG (including subclasses), IgM, and IgE antibodies in 154 paired maternal-cord serum samples from an area of meso- to hyperendemic malaria in South Cameroon. Among peripheral mother blood samples, total IgG and IgM were detected in all samples, IgE in all but two. Plasmodium falciparum-specific IgG were detected in all serum samples, IgM and IgE in > 75% of samples. The prevalence rates of anti-P. falciparum IgG subclasses varied from 75% to 97%. With the exception of P. falciparum-specific IgG, all antibody class and subclass levels were lower in cord blood than in peripheral mother blood. Plasmodium falciparum-specific IgG1 and IgG3 isotypes were transferred to the offspring more often and more efficiently than IgG2 and IgG4. The detection of total and P. falciparum-specific IgM and IgE in some cord serum samples demonstrated that fetuses can mount humoral response against malaria parasites. We also determined whether transplacentally acquired antibodies protect against malaria infection by relating the antibody levels at birth to the risk of acquiring P. falciparum infection during the first 6 months of life. Among various classes and subclasses of P. falciparum-specific antibodies, only IgG2 were related to a decrease in the risk of acquiring a P. falciparum peripheral blood infection from birth to 6 months of age.

PIP: In malaria-endemic areas, infants are relatively protected against malaria infection. Such protection is, though, to be related principally to the transplacental transfer of maternal antibodies. The authors measured total and Plasmodium falciparum-specific IgG (including subclasses), IgM, and IgE antibodies in 154 paired maternal-cord serum samples from an area of meso- to hyperendemic malaria in South Cameroon. Among maternal peripheral blood samples, total IgG and IgM were detected in all samples, IgE in all but two. P. falciparum-specific IgG antibodies were detected in all serum samples, IgM and IgE in 75% of samples. The prevalence rates of anti-P. falciparum IgG subclasses varied from 75% to 97%. With the exception of P. falciparum-specific IgG, all antibody class and subclass levels were lower in cord blood than in peripheral mother blood. P. falciparum-specific IgG1 and IgG3 isotypes were transferred to the offspring more often and more efficiently than IgG2 and IgG4. The detection of total and P. falciparum-specific IgM and IgE in some cord serum samples demonstrated that fetuses can mount humoral response against malaria parasites. The authors also determined whether transplacentally acquired antibodies protect against malaria infection by relating the antibody levels at birth to the risk of acquiring P. falciparum infection during the first 6 months of life. Among various classes and subclasses of P. falciparum-specific antibodies, only IgG2 antibodies were related to a decrease in the risk of acquiring a P. falciparum peripheral blood infection from birth to 6 months of age.

Riluzole reduces incidence of abnormal movements but not striatal cell death in a primate model of progressive striatal degeneration.

Riluzole has been shown recently to increase life expectancy in patients with amyotrophic lateral sclerosis. A number of experimental studies also suggest that this compound may be a neuroprotectant. We have investigated in baboons whether riluzole would protect striatal neurons from a prolonged 3-nitropropionic acid (3NP) treatment and ameliorate the associated motor symptoms. In animals receiving 3NP and the solvent of riluzole, 12 weeks of high-dose 3NP treatment resulted in the appearance of persistent leg dystonia and significant increases in the incidence of three categories of abnormal movements and in the dyskinesia index in the apomorphine test (0.5 mg/kg i.m.). Quantitative assessment of these behavioral deficits using a video movement analysis system demonstrated a significant decrease in locomotor activity and peak tangential velocity in 3NP-treated animals compared to controls. Histological analysis showed the presence of severe, bilateral, striatal lesions, localized in both caudate and putamen. Cotreatment with riluzole (4 mg/kg i.p., twice daily) significantly reduced the dyskinesia index (-35%, P < 0.02) in the apomorphine test. In the quantitative behavioral analysis, riluzole significantly ameliorated the decrease in peak tangential velocity (P < 0.02) but not the decrease in locomotor activity observed after 3NP. Comparative histological analysis of the two groups of treated animals did not demonstrate a clear neuroprotective effect of riluzole. The present study suggests that one potential therapeutic interest for riluzole in neurodegenerative disorders may reside in the reduction of motor symptoms associated with striatal lesions.

Maturation of fetal human neural xenografts in the adult rat brain.

Transplantation of human fetal neural cells has been used for several years as a treatment for Parkinson's disease. These therapeutic trials were based on a large number of rat allografts studies, and the species to species extrapolation appeared valid in many respects. One major difference between neurons of various species, however, is their rate of maturation; indeed, human neurons have been proven to grow much more slowly than rat neurons. This has been studied mostly, up to now, at the light microscope level. In an attempt to determine the fine structural correlates of this protracted development and to detail the schedule of morphogenesis and synaptogenesis, human fetal brain stem tissue (at 8 weeks of gestation) was transplanted into a previously lesioned brain area of immunosuppressed adult rats. Transplants, which were allowed to develop for 15 days to 3 months, were analyzed using the electron microscope. At 15 days, small cells containing a large nucleus were surrounded by wide extracellular spaces. At 1 month, grafted neurons displayed a thin rim of cytoplasm and few thin processes. At 2 months, extracellular spaces tended to diminish. Thin processes formed bundles and large processes extended from enlarged neurons. Major changes were observed at 3 months survival as the neuropile filled up with cells and processes and synaptogenesis began. Comparison with a similar ultrastructural study of thalamic rat allografts shows that human cells develop following a pattern similar to that in rat cells but that the duration of each maturation step is largely extended.

Chemical modification and site-directed mutagenesis of cysteine residues in human placental S-adenosylhomocysteine hydrolase.

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1. 1) was inactivated by 5',5-dithiobis(2-nitrobenzoic acid) following pseudo-first-order kinetics. Modification of three of the 10 cysteine residues per enzyme subunit resulted in complete inactivation of the enzyme. The three modified cysteine residues were identified as Cys113, Cys195, and Cys421, respectively, by protein sequencing after modification with [1-14C]iodoacetamide. Of the three modifiable cysteines, Cys113 and Cys195 could be protected from modification in the presence of the substrate adenosine (Ado), which also protected the enzyme from inactivation. On the other hand, Cys421 was not protected by Ado, and modification of Cys421 alone did not affect the enzyme activity. To verify whether some of these cysteine residues are important for the enzyme catalysis, these three cysteine residues were replaced by either serine or aspartic acid using site-directed mutagenesis. Mutants of both Cys113 (C113S and C113D) and Cys421 (C421S and C421D) had enzyme activities similar to that of the wild-type enzyme, and only slight changes were observed in the steady-state kinetics measured in both the synthetic and hydrolytic directions. However, mutants of Cys195 (C195D and C195S) displayed drastically reduced enzyme activities, and kcat values were only 7 and 12% of that of the wild-type enzyme, respectively, resulting in a calculated loss in binding energy (DeltaDeltaG) of approximate 1 Kcal/mol. The Cys195 mutants were capable of catalyzing both the 3'-oxidative and 5'-hydrolytic reactions, as evidenced by the reduction of E.NAD+ to NADH and formation of the 5'-hydrolytic product when incubated with (E)-5', 6'-didehydro-6'-deoxy-6'-chlorohomoadenosine at rates comparable with those catalyzed by the wild-type enzyme. However, mutations of the Cys195 severely altered the 3'-reduction potential as evidenced by the drastic reduction in the rate of [2,8-3H]Ado release from the E-NADH.[2,8-3H]3'-keto-Ado complex. Circular dichroism studies of the Cys195 mutants confirmed that the observed effects are not due to changes in secondary structure. These results suggested that the Cys195 is involved in the catalytic center and may play an important role in maintaining the 3'-reduction potential for effective release of the reaction products and regeneration of the active form (NAD+ form) of the enzyme; the Cys113 is located in or near the substrate binding site, but plays no role beneficial to the catalysis; and the Cys421 is a nonessential residue, which also explains why Cys421 does not occur in any other known AdoHcy hydrolases.

Ontogeny of human striatal DARPP-32 neurons in fetuses and following xenografting to the adult rat brain.

After a number of reports indicating positive clinical outcome of intrastriatal transplantation of fetal ventral mesencephalic tissue into patients with Parkinson's disease, the time may have come to consider the possibility of using this technique to treat patients with Huntington's disease. On the basis of the available literature, the Network of European CNS Transplantation and Restoration has established a program aiming at defining the optimal conditions for such clinical trials. The present study, conducted within this framework, pursued the goal of providing information concerning the period of striatal neuronal ontogeny in humans, taking into account the technical and legal requirements imposed by the clinical procedure of neural transplantation using human tissue. On this basis, it aimed at establishing a reliable dissecting method for the intrastriatal grafting of human fetal striatal neurons. The ontogeny of medium-spiny neurons within the developing striatum was first studied in a series of human fetal brains, 5 to 10 weeks postconception, using immunocytochemical detection of DARPP-32. Immunoreactive neurons were observed in fetuses at 7 weeks of age and older. They were mostly localized in clusters, packed in the lateral ganglionic eminence. Over a 2-week-long period, DARPP-32 neurons increased in number. Their morphology remained poorly differentiated, however, with small cell bodies, few branched dendrites, and variable intensity of immunostaining. Based on these findings, selective dissection of the lateral ganglionic eminence was carried out. This tissue was stereotaxically implanted into the striatum of immunosuppressed adult rats previously lesioned. Two months postgrafting, DARPP-32 neurons were observed as discrete patches, embedded within areas of essentially DARPP-32-negative tissue. Up to 2 months after grafting, neurons remained poorly differentiated in general, with only a few neurons exhibiting a dense immunoreactivity and long processes. These results indicate that striatal DARPP-32-immunoreactive neurons are present in the lateral ganglionic eminence in fetuses as soon as 7 weeks postconception. The striatal tissue can be dissected out and successfully transplanted. Within the grafts, neuronal differentiation appears to be a very long process, suggesting that many months might be necessary for these neurons to become functionally integrated into an adult host brain.

Developmental changes of NADPH-diaphorase neurons in the forebrain of neonatal and adult cat.

We examined morphological changes of neurons stained for NADPH-diaphorase (a marker for nitric oxide synthase, NOS) in maturing cat brains. In the newborn and 2-week-old kittens reactive neurons were dispersed throughout the cortical layers, in the white matter and in subcortical structures, with dense staining in some thalamic nuclei. In the adult, the density of reactive neurons was considerably decreased in the cortex and the white matter. In the thalamus, only some nuclei retained a faint labeling. Morphological changes also occurred at the cellular level. In the neonate, stained cells had prominent, thick processes with numerous beads and varicosities. In the adult, the processes were longer and thinner, with smaller varicosities. These observations provide further evidence that NOS may play a role during development.

Limited proteolysis of S-adenosylhomocysteine hydrolase: implications for the three-dimensional structure.

S-Adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited proteolytic digestion utilizing the proteases trypsin, V8, and papain. Results of the trypsin digest revealed one major susceptible peptide bond between amino acid residues 103 and 104 which is most likely exposed to solvent. Binding of the substrate adenosine substantially reduced the susceptibility of this site, indicating that this peptide bond may be located at or near the substrate binding site. Wild-type AdoHcy hydrolase (which exists as a tetramer) was completely resistant to V8 digestion, while a site-directed mutant form (in which Lys at position 426 was changed to Glu) of the enzyme that exists primarily as a monomer had one major V8 protease cleavage site between amino acid residues 198 and 199, suggesting that these two amino acid residues may be positioned within the tetramerization region of each subunit. Limited papain digestion of AdoHcy hydrolase revealed that the enzyme, despite multiple peptide bond cleavages, was able to maintain its quaternary structure and remain catalytically functional. This observation suggests that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding. Identification of a surface-exposed peptide bond and one located in the tetramerization domain of each subunit may provide some constraints on how each subunit can be oriented in space. Results from this study support a previously described model (D. B. Ault-Riché, C. S. Yuan, and R. T. Borchardt (1994) J. Biol. Chem., 269, 31, 472-31, 478) in which the formation of the active site is dependent upon proper quaternary structure and also suggest that the active site of the enzyme may be located at or near the tetramerization domain of each subunit.

A single mutation at lysine 426 of human placental S-adenosylhomocysteine hydrolase inactivates the enzyme.

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy), as well as the reverse reaction, through a mechanism involving an NAD(+)-dependent oxidation of the 3'-hydroxyl group of AdoHcy (3'-oxidative activity), followed by elimination of Hcy to form 3'-keto-4',5'-didehydro-5'-deoxy-Ado. The addition of water at the 5'-position (5'-hydrolytic activity) of this tightly bound intermediate, followed by an NADH-dependent reduction, results in the formation of Ado. Based on a computer graphics model of the active site of this enzyme, it was hypothesized that amino acid residues at the carboxyl-terminal end of the protein reside in the active site of the enzyme and could play a role in catalyzing the 5'-hydrolytic reaction (Yeh, J. C., Borchardt, R. T., and Vedani, A. (1991) J. Comput. Aided Mol. Des. 5, 213-234). Using site-directed mutagenesis, we show here that lysine 426 is essential for the catalytic activity of the enzyme and that it appears to play a crucial role in the 5'-hydrolytic activity and/or stability of the quaternary structure of the human placental enzyme. Mutation of Lys-426 to arginine (K426R) produces a stable tetrameric enzyme that lacks overall catalytic activity and that was isolated predominantly as its NADH form containing tightly bound 3'-keto-Ado, suggesting that the K426R mutant has oxidative activity, but lacks 5'-hydrolytic activity, preventing it from completing the entire catalytic cycle. Mutations of Lys-426 to glutamic acid (K426E) and alanine (K426A) produce enzymes that exist primarily as monomers, do not bind NAD+ or NADH, and lack catalytic activity. The results of the Lys-426 mutations suggest that this lysine residue is crucial for the 5'-hydrolytic activity of the enzyme and/or stabilizing the quaternary structure of the enzyme.

Chronic MPTP treatment reproduces in baboons the differential vulnerability of mesencephalic dopaminergic neurons observed in Parkinson's disease.

Chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to baboons was shown previously to result in a motor syndrome and a pattern of striatal dopaminergic fibre loss similar to those observed in idiopathic Parkinson's disease. In the present study, tyrosine hydroxylase-immunoreactive neurons were quantified in the mesencephalon of control (n = 4) and chronically MPTP-treated (n = 3) baboons. MPTP induced a significant reduction in neuronal cell density in the substantia nigra (63.8% reduction) and the ventral tegmental area (53.1%). Within the substantia nigra, obvious mediolateral and dorsoventral gradients of neuronal cell loss were observed. First, the pars lateralis was more affected than the lateral divisions of the pars compacta (89.6% vs 73.8% cell loss), which in turn were more depleted than the medial divisions (60.1% reduction). Second, the ventral regions of the pars compacta were more degenerated than the dorsal parts (82.4 vs 51.5% decrease). This regional pattern is strikingly similar to that observed in Parkinson's disease and indicates that two subpopulations of dopaminergic neurons are distinguishable on the basis of their differential vulnerability to MPTP. Finally, the present study confirms that chronic mitochondrial complex I inhibition using MPTP in primates is sufficient to reproduce the typical dopaminergic cell loss and striatal fibre depletion observed in Parkinson's disease.

Effects of 4'-modified analogs of aristeromycin on the metabolism of S-adenosyl-L-homocysteine in murine L929 cells.

(1'R,2'S,3')-9-(2',3'-Dihydroxycyclopentan-1'-yl)adenine (DHCaA), (1'R,2'S,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)-3-deazaadenine (3-deaza-DHCaA), (4'R)-4'-methyl-DHCaA, and (4'R)-4'-vinyl-DHCaA, which are analogs of the carbocyclic nucleoside aristeromycin, were synthesized earlier by our laboratory and were shown to be potent inhibitors of purified bovine liver S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1). In the present study, these analogs were shown to produce rapid (within 15 min) and concentration-dependent (0.03-10 microM) inhibition of AdoHcy hydrolase in cultured murine L929 cells [relative order of inhibitory activity, DHCaA = 3-deaza-DHCaA >> (4'R)-4'-vinyl-DHCaA = (4'R)-4'-methyl-DHCaA]. The relative potencies of these inhibitors on the L929 AdoHcy hydrolase were consistent with their inhibitory effects on the recombinant forms of rat liver and human placental enzymes. This inhibition of L929 cellular AdoHcy hydrolase persisted for up to 48 hr. The inhibition of the L929 AdoHcy hydrolase resulted in a significant increase in the cellular concentrations of AdoHcy, whereas the cellular S-adenosylmethionine (AdoMet) levels remained relatively constant, thereby elevating the AdoHcy/AdoMet ratios. Maximum increases in AdoHcy levels and AdoHcy/AdoMet ratios occurred within 6 hr of exposure to the inhibitors and persisted for at least 24 hr. At a concentration of 1 microM, DHCaA and 3-deaza-DHCaA increased AdoHcy/AdoMet ratios to approximately 0.8 (after 24 hr of exposure to the inhibitors), whereas (4'R)-4'-vinyl-DHCaA and (4'R)-4'-methyl-DHCaA elevated AdoHcy/AdoMet ratios to approximately 0.15, compared with control levels of 0.05. Treatment of L929 cells with concentrations of DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA up to 10 microM did not result in changes in cellular levels of endogenous nucleotides (e.g., CTP, UTP, ATP, and GTP). In contrast, cells treated with 10 microM aristeromycin for 6 hr contained reduced cellular levels of CTP, ATP, and GTP and significant levels of aristeromycin triphosphate and a GTP metabolite of this carbocyclic nucleoside. These data clearly show that the 4'-modified analogs [DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA] retain inhibitory activity toward cellular AdoHcy hydrolase, causing elevated levels of AdoHcy and elevated AdoHcy/AdoMet ratios. However, these analogs are devoid of substrate or inhibitory activity toward cellular adenosine kinase. In addition, aristeromycin is rapidly metabolized in murine L929 cell lysates, i.e., > 60% of the aristeromycin had been metabolized in 6 hr. In contrast, neither DHCaA nor 3-deaza-DHCaA showed any decrease in concentration after incubation with cell lysates for up to 6 hr.

Stable parkinsonian syndrome and uneven loss of striatal dopamine fibres following chronic MPTP administration in baboons.

The progressive degeneration of dopamine neurons observed in idiopathic Parkinson's disease was mimicked by injecting low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to baboons, on a chronic basis. Five Papio papio baboons were treated on two different regimens (chronic intravenous administration at weekly intervals for 20-21 months or, daily MPTP treatment for five days followed five to six months later by chronic weekly injections for 5-21.5 months). All animals were assessed for motor symptoms during and after neurotoxic treatment. Both regimens invariably resulted in the appearance of a progressive and irreversible syndrome characterized by action and resting tremor, cogwheel rigidity, postural impairments, hypokinesia and bradykinesia. In some animals, symptoms of resting tremor and rigidity initially restricted to one side of the body became bilateral within a few months of treatment. Subtle abnormalities that may be found in idiopathic Parkinson's disease such as alterations of the blink reflex response were also noted. Neuropathological examination of caudate nucleus, putamen, substantia nigra and ventral tegmental area in brain sections stained for tyrosine hydroxylase showed a typical uneven striatal dopamine fibre loss and a neuronal depletion in the dopaminergic mesencephalic cell groups that reproduce those observed in idiopathic Parkinson's disease. Immunocytochemical observations and behavioural data show that chronic rather than acute MPTP injection regimens can replicate most of the neuropathological and the clinical features typical of idiopathic Parkinson's disease, possibly by increasing the ability of this neurotoxin to target specific subpopulations of mesencephalic dopaminergic neurons.