Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum ‘Xanthi’) plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.     

Affinity labeling of the active site of pig liver NADH-cytochrome b5 reductase by 5′-p-fluorosulfonylbenzoyladenosine

Lysosome-solubilized pig liver NADH-cytochrome b₅ reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/K _obs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.K _d or the initial reversible enzyme-analogue complex is estimated at 185 µM withK ₂=0.22 min^?1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[¹⁴C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b₅ reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme.     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Is the Redox State of the ci Heme of the Cytochrome b6f Complex Dependent on the Occupation and Structure of the Qi Site and Vice Versa?

Oxidoreductases of the cytochrome bc₁/b₆f family transfer electrons from a liposoluble quinol to a soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. The crystal structure of cyt b₆f has revealed the presence in the Q_i site of an atypical c-type heme, heme c_i. Surprisingly, the protein does not provide any axial ligand to the iron of this heme, and its surrounding structure suggests it can be accessed by exogenous ligand. In this work we describe a mutagenesis approach aimed at characterizing the c_i heme and its interaction with the Q_i site environment. We engineered a mutant of Chlamydomonas reinhardtii in which Phe⁴⁰ from subunit IV was substituted by a tyrosine. This results in a dramatic slowing down of the reoxidation of the b hemes under single flash excitation, suggesting hindered accessibility of the heme to its quinone substrate. This modified accessibility likely originates from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation. Indeed, it also results in a marked downshift of the c_i heme midpoint potential (from +100 mV to −200 mV at pH 7). Yet the overall turnover rate of the mutant cytochrome b₆f complex under continuous illumination was found similar to the wild type one, both in vitro and in vivo. We propose that, in the mutant, a change in the ligation state of the heme upon its reduction could act as a redox switch that would control the accessibility of the substrate to the heme and trigger the catalysis.The cytochrome b₆f complex of oxygenic photosynthesis is the integral membrane protein, the quinol:plastocyanin oxido-reductase activity of which allows the linear electron flow between the two photosystems (PSI and PSII).⁴ The turnover of the cytochrome b₆f complex depends on the steady state of its redox partners, the liposoluble plastoquinol (PQH₂ reduced and protonated plastoquinone PQ) formed by the PSII, and the hydrosoluble plastocyanin oxidized by the PSI. In the Q_o site, exposed to the lumenal side, the quinol is oxidized, and this oxidation is coupled to the release of two protons into the lumen. The two electrons provided by the quinol are transferred along two bifurcated pathways, the high and low potential chains. The high potential chain involves two lumenal redox partners, the membrane-anchored flexible Rieske [2Fe-2S] cluster and the cytochrome f, which ultimately interacts with the soluble plastocyanin. In the low potential chain, electrons are transferred to the stroma via the low and high potential b hemes (b_L and b_H) of the transmembrane b₆ subunit. Two turnovers of the cyt b₆f complex lead to the reduction of the low potential chain, thereby allowing the reduction of a quinone molecule in the stromal Q_i pocket. This mechanism, which recycles reducing equivalents, is referred to as the “Q cycle,” initially described by Mitchell (1) and modified later by Crofts et al. and others (2, 3).Although this quinol:cytochrome oxidoreductase activity is involved in both the respiratory and photosynthetic electron transfer chains, recent x-ray data (4–6) have evidenced major structural differences between the b₆f complex and its mitochondrial counterpart the bc₁ complex (for reviews see Refs. 7–10). Indeed an additional heme localized in close contact with heme b_H stands as another putative electron carrier as proposed earlier by Lavergne (11). Since it was brought to light by the x-ray studies, knowledge of the basic properties of this heme, named c_i in reference to the Q_i site (5) or c_n in reference to its proximity to the negatively charged side of the membrane (4), has significantly improved. The proteins involved in the assembly machinery of the heme have been identified in Chlamydomonas reinhardtii and Arabidopsis thaliana (12, 13). Consistent with the structure, according to which the only axial ligand could be a water molecule interacting with the proponiate chain of the b_H heme, the spectroscopic properties of this heme are those of a high spin heme (14, 15). Evidences for a high spin heme covalently bound to the cytochrome b subunit were also found in Heliobacterium modesticaldum and Heliobacillus mobilis (16).In the b₆f complex from the oxygenic photosynthetic chain, EPR (15) and structural data (17) have shown that NQNO (2-n-nony l-4-hydroxyquinoline N-oxide), an inhibitor of the Q_i pocket (18, 19), can act as an axial ligand to c_i. This ligation is accompanied by a significant change in the redox properties of c_i, because, in the presence of NQNO, at least two titrations waves were observed (13, 14), one with a midpoint potential (E_m) similar to that observed in the absence of NQNO and the other with a midpoint potential downshifted by ∼250 mV. This, together with the widespread range of redox potential found for heme c_i (11, 14, 15, 20), points to a structural plasticity of the c_i ligand network.This plasticity may arise from the unusual coordination properties of the heme c_i. As a matter of fact, the x-ray models of the complex from C. reinhardtii and Mastigocladus laminosus evidenced a water or hydroxyl molecule as a fifth ligand. The sixth position of coordination is directed toward the Q_i pocket and appears as free. Nevertheless, the side chain of Phe⁴⁰ of subunit IV protrudes above the heme plane, leaving little space for any axial ligand to the heme c_i. Besides, modeling a quinone analogue in the electron density found in the Q_i pocket of structures obtained in presence of Tridecyl- Stigmatellin or NQNO implies a steric clash with the native position of the Phe⁴⁰ aromatic ring.⁵ The Phe⁴⁰ residue of subunit IV therefore stands as a key residue for the plasticity of the site, making it an ideal mutagenesis target when attempting to alter the possible interactions between c_i and the quinone or quinol (4, 5) (Fig. 1). Here we present the consequences of the substitution of Phe⁴⁰ by a tyrosine on the properties and function of the c_i heme.Open in a separate windowFIGURE 1.A view of the Q_i site from the dimer interface. The coordinates are from the Protein Data Bank 1Q90 model. The Van der Waal''s surface of the peptide chains was drawn with Pymol. Phe⁴⁰ is in van der Waal''s contact with the plane of the c_i, heme.     

Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabissativa

?1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ?1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75? resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.     

Structural and Functional Elements of the Promoter Encoded by the 5′ Untranslated Region of the Venezuelan Equine Encephalitis Virus Genome

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. The pathogenesis of this virus depends strongly on the sequences of the structural proteins and on the mutations in the RNA promoter encoded by the 5′ untranslated region (5′UTR) of the viral genome. In this study, we performed a detailed investigation of the structural and functional elements of the 5′-terminal promoter and analyzed the effect of multiple mutations introduced into the VEEV 5′UTR on virus and RNA replication. The results of this study demonstrate that RNA replication is determined by two synergistically functioning RNA elements. One of them is a very 5′-terminal AU dinucleotide, which is not involved in the stable RNA secondary structure, and the second is a short, G-C-rich RNA stem. An increase or decrease in the stem''s stability has deleterious effects on virus and RNA replication. In response to mutations in these RNA elements, VEEV replicative machinery was capable of developing new, compensatory sequences in the 5′UTR either containing 5′-terminal AUG or AU repeats or leading to the formation of new, heterologous stem-loops. Analysis of the numerous compensatory mutations suggested that at least two different mechanisms are involved in their generation. Some of the modifications introduced into the 5′ terminus of the viral genome led to an accumulation of the mutations in the VEEV nsPs, which suggested to us that there is a direct involvement of these proteins in promoter recognition. Furthermore, our data provide new evidence that the 3′ terminus of the negative-strand viral genome in the double-stranded RNA replicative intermediate is represented by a single-stranded RNA. Both the overall folding and the sequence determine its efficient function as a promoter for VEEV positive-strand RNA genome synthesis.Alphaviruses are a group of important human and animal pathogens. They are widely distributed both in the New and the Old Worlds and circulate between mosquito vectors and vertebrate hosts (45). In mosquitoes, they cause a persistent, life-long infection characterized by virus accumulation in salivary glands, which is required for infecting vertebrate hosts during a blood meal (50). In vertebrates, alphaviruses develop high-titer viremia, and their replication induces a variety of diseases with symptoms depending on both the host and the causative virus (11). Venezuelan equine encephalitis virus (VEEV), the New World alphavirus, is one of the most pathogenic members of the genus (16, 45). Representatives of the VEEV serocomplex circulate in Central, South, and North America and cause severe, and sometimes fatal, encephalitis in humans and horses (3, 11, 16, 24). Accordingly, VEEV represents a serious public health threat in the United States (39, 48, 51, 53), and during VEEV epizootics, equine mortality can reach 83%, and in humans, neurological diseases can be detected in up to 14% of all infected individuals, especially children (15). The overall mortality rate for humans is below 1%, but it is usually higher among children, the elderly, and, most likely, immunocompromised individuals (49). In spite of the continuous threat of VEEV epidemics, the biology of this virus, its pathogenesis, and the mechanism of replication are insufficiently understood. To date, no safe and efficient vaccine and therapeutic means have been developed for this pathogen.The VEEV genome is represented by a single-stranded, almost 11.5-kb-long RNA molecule of positive polarity. This RNA mimics the structure of cellular mRNAs by containing a cap at the 5′ ends and a poly(A) tail at the 3′ ends of the genome (18). The genomic RNA encodes two polyproteins: the 5′-terminal open reading frame (ORF) is translated into viral nonstructural proteins (nsP1 to nsP4), forming the replication enzyme complex (RC). The second ORF corresponds to the 3′-terminal one-third of the genome and encodes all of the viral structural proteins, C, E2, and E1. The latter proteins are translated from the subgenomic RNA synthesized during virus replication (45).The replication of the alphavirus genome is a highly regulated, multistep process, which includes the synthesis of three different RNA species (45). The regulation of their synthesis is achieved by differential processing of viral nsPs (22, 23, 43). First, the initially synthesized nonstructural polyprotein is partially processed by the nsP2-associated protease into P123 and nsP4, and this complex is active in negative-strand RNA synthesis (22). The latter RNA is present in the double-stranded RNA (dsRNA) replicative intermediate and is associated with plasma membrane and endosome-like vesicular organelles (8). Further processing of the polyproteins into individual nsP1 to nsP4 makes the RC capable of the synthesis of the positive-strand genome and subgenomic RNA but not of negative-strand RNA (23, 41, 42). Thus, the completely processed nsPs utilize only the promoters located on the negative strand of the viral genome.The defined promoters in the alphavirus genomes include (i) a 3′-terminal 19-nucleotide (nt)-long, conserved sequence element (CSE) adjacent to the poly(A) tail (12, 13, 19); (ii) the subgenomic promoter in the negative-strand copy of the viral genome (25); and (iii) the promoter for the synthesis of the positive-strand viral genome (45). The latter promoter is located at the 3′ end of the negative strand of the viral genome and has a complex structure. The two identified elements include the sequence, encoded by the 5′ untranslated region (5′UTR) (a core promoter) (5, 9, 32), and a 51-nt CSE, found ∼150 nt downstream of the genome''s 5′ terminus in the nsP1-encoding sequence. Our previous results and those of other research groups demonstrated that the 51-nt CSE functions as a replication enhancer in a virus- and cell-dependent mode (4, 33). Clustered mutations in the VEEV 51-nt CSE or its complete deletion either had deleterious effects on RNA replication or completely abolished RNA synthesis (30). However, RNA replication was ultimately recovered due to an accumulation of compensatory, adaptive mutations in either VEEV nsP2 or nsP3 (30). Thus, the 51-nt CSE in the VEEV genome is not absolutely essential for virus replication, but its presence is highly beneficial for achieving the most efficient growth rates in cells of both vertebrate and invertebrate origins. Alphavirus core promoters demonstrate a very low level of sequence conservation and also function in cell- and virus-specific modes (9). Previous studies suggested that the sequence and/or secondary structure of the VEEV core promoter plays a critical role in virus pathogenesis, and the G3→A (A3) mutation, found in an attenuated strain of VEEV TC-83, is one of the determinants of its less pathogenic phenotype (17, 55). However, information about functional elements of the VEEV core promoter remains incomplete, and its structural and functional elements have not yet been dissected.In this study, we applied a combination of molecular approaches to further define the functional components of the VEEV 5′UTR-specific core promoter, which mediates positive-strand genome synthesis. Our results demonstrate the presence of three structural RNA elements, two of which synergistically determine promoter activity. The first element of the promoter is a very short, 5′-terminal sequence, which is not involved in a stable secondary structure. Point mutations in the very 5′-terminal nucleotides have a deleterious effect on genome RNA replication. The second element is the short RNA stem, located in close proximity to the 5′ end of the genome. Mutations changing either the stability or sequence of the stem strongly affect virus replication and cause its rapid evolution, leading to the appearance of heterologous repeating elements in the unpaired 5′ terminus or the generation of other sequences that might potentially fold into stem structures. Surprisingly, the third structural RNA element, the loop, appears to play no important role in RNA replication and can be replaced either by a shorter loop or by the loop having a heterologous sequence without a detectable effect on virus and RNA replication.     

Analysis of the Gut Microbiota by High-Throughput Sequencing of the V5–V6 Regions of the 16S rRNA Gene in Donkey

Considerable evidence suggests that the gut microbiota is complex in many mammals and gut bacteria communities are essential for maintaining gut homeostasis. To date the research on the gut microbiota of donkey is surprisingly scarce. Therefore, we performed high-throughput sequencing of the 16S rRNA genes V5–V6 hypervariable regions from gut fecal material to characterize the gut microbiota of healthy donkeys and compare the difference of gut microbiota between male and female donkeys. Sixty healthy donkeys (30 males and 30 females) were enrolled in the study, a total of 915,691 validated reads were obtained, and the bacteria found belonged to 21 phyla and 183 genera. At the phylum level, the bacterial community composition was similar for the male and female donkeys and predominated by Firmicutes (64 % males and 64 % females) and Bacteroidetes (23 % males and 21 % females), followed by Verrucomicrobia, Euryarchaeota, Spirochaetes, and Proteobacteria. At the genus level, Akkermansia was the most abundant genus (23 % males and 17 % females), followed by Sporobacter, Methanobrevibacter, and Treponema, detected in higher distribution proportion in males than in females. On the contrary, Acinetobacter and Lysinibacillus were lower in males than in females. In addition, six phyla and 15 genera were significantly different between the male and female donkeys for species abundance. These findings provide previously unknown information about the gut microbiota of donkeys and also provide a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in donkey and other animals.     

Modification of secondary head-forming activity of microinjected ∆β-catenin mRNA by co-injected spermine and spermidine in Xenopus early embryos

Polyamines are essential for cell growth and differentiation. In Xenopus early embryos, per embryo level of spermine is extremely low as compared with that of spermidine. To disclose the possible function of polyamines in Xenopus early embryos, we tested the effect of co-injection of spermine and spermidine on the functioning of exogenously microinjected in vitro-synthesized, ?β-catenin mRNA which is known to induce a secondary head after being microinjected into a ventral vegetal blastomere in 8-celled Xenopus embryos. Microinjection of ?β-catenin mRNA in fact induced a secondary axis with a secondary head, and co-injection of spermine or spermidine suppresses induction of the secondary head and secondary axis without drastic effects like induction of immediate cell death or execution of apoptosis at blastula stage. The inhibitory effects were dosage dependent, and at lower doses the inhibition was mainly on secondary head formation rather than on secondary axis formation. We performed similar experiments using GFP mRNA and confirmed that expression of GFP mRNA was also suppressed by co-injection of spermine. We mixed ?β-catenin mRNA with different amounts of spermine and performed electrophoresis on agarose gels, with a finding that the prior mixing greatly suppressed mRNA migration. These results suggest that an excess amount of spermine as well as spermidine exerts inhibitory effects on mRNA translation, and that the inhibition may be due to direct binding of polyamines to mRNA and a reduction of negative charges on mRNA molecules that might also induce the formation of cross link-like networks among mRNAs.     

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum

Bax?2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax?2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax?2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax?2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax?2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax?2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of Bax?2 in Purkinje cells.     

Exfoliation of the β-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed ‘exosomes’ into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, G_s, and the inhibitory, G_i, regulatory components) and β-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of G_s or G_i in the exosomes was detected by ADP ribosylation using [α-³²P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal G_s was almost as active as the membrane-derived G_s in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc⁻ cells, which lack a functional G_s. The ability of exosomal G_s to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane G_s. The density of β-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated β-adrenergic receptors seem to be impaired in their ability to couple to and activate G_s. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the β-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.     

Ifenprodil Attenuates Methamphetamine-Induced Behavioral Sensitization and Activation of Ras-ERK-∆FosB Pathway in the Caudate Putamen

Addiction is a debilitating, chronic psychiatric disorder that is difficult to cure completely owing to the high rate of relapse. Behavioral sensitization is considered to may underlie behavioral changes, such as relapse, caused by chronic abuse of psychomotor stimulants. Thus, its animal models have been widely used to explore the etiology of addiction. Recently, increasing evidence has demonstrated that N-methyl-d-aspartate receptors (NMDARs) play an important role in addiction to psychomotor stimulants. However, the role of GluN2B-containing receptors and their downstream signaling pathway(s) in behavioral sensitization induced by methamphetamine (METH) have not been investigated yet. In this study, we used different doses of ifenprodil (2.5, 5, 10 mg/kg), a selective antagonist of the GluN2B subunit, to investigate the role of GluN2B-containing NMDARs in METH-induced behavioral sensitization. We then examined changes in the levels of Ras, phosphorylated extracellular signal-regulated kinase (pERK)/ERK, and ?FosB in the caudate putamen (CPu) by western blot. We found that 2.5 or 10 mg/kg ifenprodil significantly attenuated METH-induced behavioral sensitization, whereas the mice treated with a moderate dose of ifenprodil (5 mg/kg) displayed no significant changes. Further results of western blot experiments showed that repeated administration of METH caused the increases in the levels of Ras, pERK/ERK and ?FosB in the CPu, and these changes were inhibited by only the 2.5 mg/kg dose of ifenprodil. In conclusion, these results demonstrated that 2.5 mg/kg ifenprodil could attenuate METH-induced behavioral sensitization. Moreover, GluN2B-containing NMDARs and their downstream Ras-ERK-?FosB signaling pathway in the CPu might be involved in METH-induced behavioral sensitization.     

Phylogeny and Evolution of Selected Primates as Determined by Sequences of the ε-Globin Locus and 5′ Flanking Regions

We studied phylogenetic relationships of 39 primate species using sequences of the -globin gene. For 13 species, we also included flanking sequences 5 of this locus. Parsimony analyses support the association of tarsiers with the anthropoids. Our analysis of New World monkeys supports the model in which the callitrichines form a clade with Aotus, Cebus, and Saimiri, with Cebus and Saimiri being sister taxa. However, analysis of the 5 flanking sequences did not support grouping the atelines with Callicebus and the pitheciins. Our data support the classification of platyrrhines into three families, Cebidae (consisting of Cebus, Saimiri, Aotus, and the callitrichines; Atelidae—the atelines; and Pitheciidae—Callicebus and the pithiciins. The strepsirhines form well-defined lemuroid and lorisoid clades, with the cheirogaleids (dwarf and mouse lemurs) and Daubentonia (aye-aye) in the lemuroids, and the aye-aye being the most anciently derived. These results support the hypothesis that nonhuman primates of Madagascar descended from a single lineage. Local molecular clock calculations indicate that the divergence of lemuroid and lorisoid lineages, and the earliest diversification of lemuroids, occurred during the Eocene. The divergence of major lorisoid lineages was probably considerably more recent, possibly near the Miocene–Oligocene boundary. Within hominoids some estimated dates differ somewhat from those found with more extensive noncoding sequences in the -globin cluster.