Formate-producing capacity provided by reducing ability of Streptococcus thermophilus nicotinamide adenine dinucleotide oxidase determines yogurt acidification rate.

Yogurt is made by fermenting milk with 2 lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. To comprehensively understand the protocooperation mechanism between S. thermophilus and L. bulgaricus in yogurt fermentation, we examined 24 combinations of cocultures comprising 7 fast- or slow-acidifying S. thermophilus strains with 6 fast- or slow-acidifying L. bulgaricus strains. Furthermore, 3 NADH oxidase (Nox)-deficient mutants (Δnox) and one pyruvate formate-lyase deficient mutant (ΔpflB) of S. thermophilus were used to evaluate the factor that determines the acidification rate of S. thermophilus. The results revealed that the acidification rate of S. thermophilus monoculture determined the yogurt fermentation rates, despite the coexistence of L. bulgaricus, whose acidification rate was either fast or slow. Significant correlation was found between the acidification rate of S. thermophilus monoculture and the amount of formate production. Result using ΔpflB showed that the formate was indispensable for the acidification of S. thermophilus. Moreover, results of the Δnox experiments revealed that formate production required Nox activity, which not only regulated dissolved oxygen, but also the redox potential. The Nox provided the large decrease in redox potential required by pyruvate formate-lyase to produce formate. A highly significant correlation was found between formate accumulation and Nox activity in S. thermophilus. In conclusion, the formate production ability provided by the action of Nox activity determines the acidification rate of S. thermophilus, and consequently, regulates yogurt coculture fermentation.

Hippocampal cells encode places by forming small anatomical clusters.

The hippocampus has been hypothesized to function as a "spatial" or "cognitive" map, however, the functional cellular organization of the spatial map remains a mystery. The majority of electrophysiological studies, thus far, have supported the view of a random-type organization in the hippocampus. However, using immediate early genes (IEGs) as an indicator of neuronal activity, we recently observed a cluster-type organization of hippocampal principal cells, whereby a small number ( approximately 4) of nearby cells were activated in rats exposed to a restricted part of an environment. To determine the fine structure of these clusters and to provide a 3D image of active hippocampal cells that encode for different parts of an environment, we established a functional mapping of IEGs zif268 and Homer1a, using in situ hybridization and 3D-reconstruction imaging methods. We found that, in rats exposed to the same location twice, there were significantly more double IEG-expressing cells, and the clusters of nearby cells were more "tightly" formed, in comparison to rats exposed to two different locations. We propose that spatial encoding recruits specific cell ensembles in the hippocampus and that with repeated exposure to the same place the ensembles become better organized to more accurately represent the "spatial map."

Estrogen and aging affect synaptic distribution of phosphorylated LIM kinase (pLIMK) in CA1 region of female rat hippocampus.

17beta-Estradiol (E) increases axospinous synapse density in the hippocampal CA1 region of young female rats, but not in aged rats. This may be linked to age-related alterations in signaling pathways activated by synaptic estrogen receptor alpha (ER-alpha) that potentially regulate spine formation, such as LIM-kinase (LIMK), an actin depolymerizing factor/cofilin kinase. We hypothesized that, as with ER-alpha, phospho-LIM-kinase (pLIMK) may be less abundant or responsive to E in CA1 synapses of aged female rats. To address this, cellular and subcellular distribution of pLIMK-immunoreactivity (IR) in CA1 was analyzed by light and electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle or E. pLIMK-IR was found primarily in perikarya within the pyramidal cell layer and dendritic shafts and spines in stratum radiatum (SR). While pLIMK-IR was occasionally present in terminals, post-embedding quantitative analysis of SR showed that pLIMK had a predominant post-synaptic localization and was preferentially localized within the postsynaptic density (PSD). The percentage of pLIMK-labeled synapses increased (30%) with E treatment (P<0.02) in young animals, and decreased (43%) with age (P<0.002) regardless of treatment. The pattern of distribution of pLIMK-IR within dendritic spines and synapses was unaffected by age or E treatment, with the exception of an E-induced increase in the non-synaptic core of spines in young females. These data suggest that age-related synaptic alterations similar to those seen with ER-alpha occur with signaling molecules such as pLIMK, and support the hypothesis that age-related failure of E treatment to increase synapse number in CA1 may be due to changes in the molecular profile of axospinous synapses with respect to signaling pathways linked to formation of additional spines and synapses in response to E.

Differential distribution of estrogen receptor (ER)-alpha and ER-beta in the midbrain raphe nuclei and periaqueductal gray in male mouse: Predominant role of ER-beta in midbrain serotonergic systems.

We examined the distribution of estrogen receptor (ER)-alpha and ER-beta immunoreactive (ir) cells in the dorsal (DRN) and median/paramedian (MPRN) raphe nuclei in male mice. ER-alpha ir neurons were scattered across the three subdivisions (ventral, dorsal, and lateral) of the DRN and the MPRN. Robust ER-beta ir cells were observed throughout the raphe nuclei, and were particularly abundant in the ventral and dorsal subdivisions of the DRN. Using dual-label immunocytochemistry for ER-alpha or ER-beta with tryptophan hydroxylase (TPH), the rate-limiting enzyme for 5-hydroxytryptamine (5-HT) synthesis, over 90% of ER-beta ir cells exhibited TPH-ir in all DRN subdivisions, whereas only 23% of ER-alpha ir cells contained TPH. Comparisons of ER-alpha knockout (alphaERKO) as well as ER-beta knockout (betaERKO) mice with their respective wild-type (WT) littermates revealed that gene disruption of either ER-alpha or ER-beta did not affect the other ER subtype expression in the raphe nuclei. In situ hybridization histochemistry revealed that there was a small but statistically significant decrease in TPH mRNA expression in the ventral DRN subdivision in betaERKO mice compared with betaWT mice, whereas TPH mRNA levels were not affected in alphaERKO mice. These findings support a hypothesis that ER-beta activation may contribute to the estrogenic regulation of neuroendocrine and behavioral functions, in part, by acting directly on 5-HT neurons in the raphe nuclei in male mice.

Spatiotemporal distribution of gp130 cytokines and their receptors after status epilepticus: comparison with neuronal degeneration and microglial activation.

Although numerous studies have demonstrated the neurotrophic capacity of gp130 cytokines, it remains unclear whether endogenously expressed cytokines actually function in a direct neuromodulatory manner. Therefore, using the lithium-pilocarpine status epilepticus model, we performed a detailed in situ hybridization time-course study of five gp130 cytokines (interleukin [IL]-6, leukemia inhibitory factor [LIF], IL-11, oncostatin-m [OSM], and ciliary neurotrophic factor), gp130, and the receptors of the cytokines we found to be induced (IL-6 receptor [IL-6R], LIF receptor [LIF-R], and IL-11 receptor [IL-11R]). Additionally, to further understand the regulation of these cytokines, we compared their expression with the pattern of neuronal degeneration and microglial activation. Under control conditions, all cytokines, except LIF, exhibited faint to moderate expression in hippocampal principal layers. After seizure, IL-6, LIF, and IL-11 exhibited a rapid, robust, and transient upregulation in non-principal cells. LIF also exhibited a remarkably early and transient induction in the granule cell layer of the dentate gyrus. OSM exhibited only a mild and inconsistent induction. All receptors examined were strongly expressed only in hippocampal principal layers under control conditions. A mild and late induction of the IL-6R, LIF-R, and IL-11R occurred after seizure with a scattered distribution. A progressive and chronic induction of gp130 was observed in cells that appeared to be associated with blood vessels. Degeneration of hilar interneurons and CA1 pyramidal cells was early and progressive. Granule neurons of the dentate gyrus, however, exhibited a delayed and precipitous pattern of degeneration, specifically in the lateral portion of the superior blade. Microglial activation was maximal 24-48 h post-seizure. We speculate that gp130 cytokines play a paracrine, neuromodulatory role in the hippocampus since both before and after seizure, principal cells appear to be the major cell type expressing the receptors for these cytokines. Furthermore, we suggest that activity-dependent mechanisms may be involved in the regulation of cytokines expressed early, and that relatively late occurring cytokine expression may be elicited by injury-related stimuli.

Differential expression of suppressors of cytokine signaling-1, -2, and -3 in the rat hippocampus after seizure: implications for neuromodulation by gp130 cytokines.

Numerous studies have investigated the expression of various cytokine families in the CNS after brain injury. The gp130 or interleukin (IL)-6-type cytokines have received a great deal of focus, and it is clear that they exhibit an acute and robust upregulation in various brain injury models. We are interested to determine, however, whether endogenously expressed cytokines in the CNS act in a direct neuromodulatory manner. In an accompanying study, we examined the expression of five gp130 cytokines and their receptors in the lithium-pilocarpine model of status epilepticus. We follow up that study here by trying to determine if gp130 signal transduction occurs in hippocampal principal neurons after seizure. Therefore, using the expression of suppressors of cytokine signaling (SOCS)-1 and -3 as indices of gp130 signal transduction, we performed a detailed in situ hybridization seizure time-course study in the adult rat hippocampus. For comparison, we also examined SOCS-2, which is involved in insulin-like growth factor signaling. We found that while SOCS-1 and -3 were faintly expressed under basal conditions, only SOCS-3 exhibited a rapid, robust, and transient induction. This occurred first in non-principal cells, which appeared to be glial, peaking at approximately 12 h post-seizure. Subsequently, a robust induction of SOCS-3 occurred in pyramidal and granule neurons, peaking at approximately 24 h. SOCS-2 displayed a relatively higher level of basal expression, particularly in CA3, and a mild and transient downregulation by 24 h. These findings corroborate the hypothesis that seizure-induced gp130 cytokines play a direct neuromodulatory role in the hippocampus. Since in our previous study we did not detect cytokine receptor expression in non-principal cells, it is unclear what elicits SOCS-3 expression in this population.

Mechanism of glial activation by S100B: involvement of the transcription factor NFkappaB.

Compelling evidence links chronic activation of glia and the subsequent cycle of neuroinflammation and neuronal dysfunction to the progression of neurodegeneration in disorders such as Alzheimer's disease (AD). S100B, a glial-derived cytokine, is significantly elevated in the brains of AD patients and high concentrations of S100B are believed to be detrimental to brain function. As a first step toward elucidating the mechanisms by which S100B might be serving this detrimental role, we examined the mechanisms by which S100B stimulates glial inducible nitric oxide synthase (iNOS), an oxidative stress related enzyme that has been linked to neuropathology through the production of neurotoxic peroxynitrite. We report here that S100B stimulates iNOS in rat primary cortical astrocytes through a signal transduction pathway that involves activation of the transcription factor NFkappaB. NFkappaB activation was demonstrated by nuclear translocation of the p65 NFkappaB subunit, stimulation of NFkappaB-specific DNA binding activity, and stimulation of NFkappaB-dependent transcriptional activity. Furthermore, S100B-induced iNOS promoter activation was inhibited upon mutation of the NFkappaB response element in the promoter, and transfection of cells with an NFkappaB inhibitor blocked S100B-induced iNOS promoter activation and nitric oxide production. These studies define a signal transduction pathway by which S100B activation of glia could participate in the generation of oxidative stress in the brain.

Cerebrospinal fluid S100B is elevated in the earlier stages of Alzheimer's disease.

Postmortem demonstration of increased expression of biologically active S100B in Alzheimer's disease (AD) and its relation to progression of neuropathological changes across the cortical regions suggests involvement of this astrocytic cytokine in the pathophysiology of AD. The hypothesis that the overexpression of S100B in Alzheimer brain is related to the progression of clinical symptoms was addressed in living persons by measuring S100B concentrations in cerebrospinal fluid (CSF) from AD patients with a broad range of clinical dementia severity and from healthy older persons. The effect of normal aging on CSF S100B concentrations also was estimated. CSF S100B did not differ between all 68 AD subjects (0.98+/-0.09 ng/ml (mean+/-S.E.M.)) and 25 healthy older subjects (0.81+/-0.13 ng/ml). When AD subjects were divided into mild/moderate stage and advanced stage clinical dementia severity by the established Clinical Dementia Rating Scale (CDR) criteria, S100B was significantly higher in the 46 mild/moderate stage AD subjects (1.17+/-0.11 ng/ml) than in either the 22 advanced stage AD subjects (0.60+/-0.12 ng/ml) or the healthy older subjects. Consistent with higher CSF S100B in mild to moderate AD, there was a significant correlation among all AD subjects between CSF S100B and cognitive status as measured by the Mini Mental State Exam (MMSE) score. CSF S100B did not differ between healthy older subjects and healthy young subjects. These results suggest increased CNS expression of S100B in the earlier stages of AD, and are consistent with a role for S100B in the initiation and/or facilitation of neuritic plaque formation in AD brain.

EXAFS analysis of the zinc-binding domain of boar spermatidal transition protein 2.

Boar Spermatidal Transition Protein 2 (TP2; 137 amino acid residues) is supposed to play an important role in initiation of chromatin condensation and cessation of transcriptional activity during mammalian spermniogenesis. Boar TP2 has three potential zinc finger motifs and binds three atoms of zinc per molecule. However the structure of the zinc-binding domain of boar TP2 has not been completely determined. To elucidate the local structure around the zinc atoms of boar TP2, we performed an X-ray absorption fine structure (XAFS) measurement on the zinc-binding domain of TP2(TP2Z)(residues 1-103) in the fluorescence mode. By EXAFS analyses we have demonstrated that each of the three zinc atoms is coordinated by approximately two sulfur and two nitrogen atoms on average. The average Zn-S and Zn-N distances were found to be 2.36 and 2.01 A, respectively. The sulfur and nitrogen atoms are attributed to cysteine and histidine residues, respectively, from comparison of the EXAFS spectra with model compounds ZnS and ZnTPP zinc(II) tetraphenylporphyrin).

Tracking the estrogen receptor in neurons: implications for estrogen-induced synapse formation.

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERalpha) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERalpha and ERbeta mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERalpha-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERalpha immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Rice (Oryza sativa) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus.

We have isolated full-length cDNAs for two distinct isoforms of glutamate decarboxylase (GAD), designated OsGAD1 and OsGAD2 from a rice shoot cDNA library. Open reading frames found in OsGAD1 and OsGAD2 cDNAs encode putative proteins of 501 (56.7 kDa) and 500 amino acids (55.6 kDa), respectively. They show 69% identity to each other and 67-78% identity to dicotyledonous counterpart sequences determined so far. Comparative analysis of relevant genomic clones obtained from the rice genomic library with these cDNAs as probes demonstrated that the number and sizes of introns deduced for these two genes differ considerably. Interestingly, in the regions in the putative gene products corresponding to the C-terminal 30-amino-acid peptide known as the calmodulin-binding domain of plant GADs, OsGAD1 possesses a typical motif, while OsGAD2 contains several substitutions of amino acids that contribute strongly to the binding of calmodulin (CaM). An in vitro CaM-binding assay of these proteins over-expressed in Escherichia coli revealed that OsGAD1 can in fact bind specifically to bovine CaM but OsGAD2 cannot. RNA analysis showed that transcripts of OsGAD1 and OsGAD2 were present in all tissues examined, but their expression was differentially regulated, at least in roots and maturing seeds.

A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors.

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.

Splicing of arabidopsis tRNA(Met) precursors in tobacco cell and wheat germ extracts.

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA(GpsiA(Tyr)) and elongator tRNA(CmAU(Met)) contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA(Tyr). Here we have studied the expression of an Arabidopsis elongator tRNA(Met) gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA(Met) precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA(Met) to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3' and 5' splice sites and of a structured intron for pre-tRNA(Met) splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA(Met) splicing and that a highly structured intron is indispensable for pre-tRNA(Met) splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA(Met) gene, is efficiently processed and spliced in both plant extracts.

Identification of two catalytic subunits of tRNA splicing endonuclease from Arabidopsis thaliana.

tRNA splicing endonuclease is essential for the correct removal of introns from precursor tRNA molecules of Archaea and Eucarya. The only well-characterized eucaryotic enzyme until now is the endonuclease from yeast (Saccharomyces cerevisiae). This protein has a heterotetrameric structure. Two of the four subunits, i.e. Sen34 and Sen44, contain the active sites for cleavage at the 3'- and 5'-splice sites, respectively. We have identified three novel genes from Arabidopsis thaliana, encoding putative subunits of tRNA splicing endonuclease. They are designated as AtSen1, AtSen2, and AtpsSen1. Both genes AtSen1 and AtSen2 seem to be functionally active, as deduced from corresponding cDNA sequences. Comparison of the amino acid sequences of the these two Arabidopsis proteins revealed 72% identity. However, AtpsSen1 is more similar to AtSen1, but is very likely a pseudogene, as concluded from extended stretches of deletions and the presence of in-frame stop codons. All putative proteins contain a conserved domain at their C-terminus common to counterparts from other organisms. Interestingly, they are more similar to the yeast catalytic subunit Sen44 than to Sen34. Southern analysis with various probes revealed that each gene is present as single copies in the nuclear genome. The evolutionary implications of these findings are discussed.

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes.

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.

Beta-amyloid stimulation of inducible nitric-oxide synthase in astrocytes is interleukin-1beta- and tumor necrosis factor-alpha (TNFalpha)-dependent, and involves a TNFalpha receptor-associated factor- and NFkappaB-inducing kinase-dependent signaling mechanism.

In Alzheimer's disease, beta-amyloid (Abeta) plaques are surrounded by activated astrocytes and microglia. A growing body of evidence suggests that these activated glia contribute to neurotoxicity through the induction of inflammatory cytokines such as interleukin (IL)-1beta and tumor necrosis factor-alpha (TNFalpha) and the production of neurotoxic free radicals, mediated in part by the expression of inducible nitric-oxide synthase (iNOS). Here, we address the possibility that Abeta-stimulated iNOS expression might result from an initial induction of IL-1beta and TNFalpha. We find that in Abeta-stimulated astrocyte cultures, IL-1beta and TNFalpha production occur before iNOS production, new protein synthesis is required for increased iNOS mRNA levels, and the IL-1 receptor antagonist IL-1ra can inhibit nitrite accumulation. Likewise, dominant-negative mutants of tumor necrosis factor-alpha receptor-associated factor (TRAF) 6, TRAF2, and NFkappaB-inducing kinase (NIK), intracellular proteins involved in IL-1 and TNFalpha receptor signaling cascades, inhibit Abeta-stimulated iNOS promoter activity. Our data suggest that Abeta stimulation of astrocyte iNOS is mediated in part by IL-1beta and TNFalpha, and involves a TRAF6-, TRAF2-, and NIK-dependent signaling mechanism.

Selective modulation of BV-2 microglial activation by prostaglandin E(2). Differential effects on endotoxin-stimulated cytokine induction.

The influence of prostaglandins on glial functions and, more specifically, on glial activation is not well understood. We report here that prostaglandin E(2) (PGE(2)), one of the major prostaglandins produced in the brain, acts as a potent and selective inhibitor of tumor necrosis factor alpha (TNF-alpha) production in lipopolysaccharide-stimulated primary microglia and the microglial cell line BV-2. The IC(50) for this effect is 1 nM, and 100 nM PGE(2) suppresses TNF-alpha production by >95%. More detailed studies of BV-2 cells show that PGE(2) also prevents the secretion of interleukin (IL)-6 but does not significantly modify lipopolysaccharide-stimulated expression of cyclooxygenase-2, pro-IL-1beta, or inducible nitric oxide synthase. PGE(2) appears to act primarily at the level of translation or protein stability, because TNF-alpha and IL-6 mRNA levels were only modestly decreased at high PGE(2) concentrations; concomitantly with this inhibition, PGE(2) up-regulated the levels of IL-1beta mRNA. The effects of PGE(2) could be largely mimicked by 8-bromo-cAMP, suggesting that, as in other cell types, PGE(2) action is mediated at least in part by a rise in intracellular cyclic AMP. However, the protein kinase A inhibitor H89 only partially reversed the inhibition of TNF-alpha production by PGE(2), implying that the PGE(2) effect in BV-2 cells is mediated through both protein kinase A-dependent and -independent pathways.

Expression of a zinc-binding domain of boar spermatidal transition protein 2 in Escherichia coli.

Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 34 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1-103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO(-)(3) and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc.

Cyclopentenone prostaglandins suppress activation of microglia: down-regulation of inducible nitric-oxide synthase by 15-deoxy-Delta12,14-prostaglandin J2.

Mechanisms leading to down-regulation of activated microglia and astrocytes are poorly understood, in spite of the potentially detrimental role of activated glia in neurodegeneration. Prostaglandins, produced both by neurons and glia, may serve as mediators of glial and neuronal functions. We examined the influence of cyclopentenone prostaglandins and their precursors on activated glia. As models of glial activation, production of inducible nitric-oxide synthase (iNOS) was studied in lipopolysaccharide-stimulated rat microglia, a murine microglial cell line BV-2, and IL-1beta-stimulated rat astrocytes. Cyclopentenone prostaglandins were potent inhibitors of iNOS induction and were more effective than their precursors, prostaglandins E2 and D2. 15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) was the most potent prostaglandin among those tested. In activated microglia, 15d-PGJ2 suppressed iNOS promoter activity, iNOS mRNA, and protein levels. The action of 15d-PGJ2 does not appear to involve its nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) because troglitazone, a specific ligand of PPARgamma, was unable to inhibit iNOS induction, and neither troglitazone nor 15d-PGJ2 could stimulate the activity of a PPAR-dependent promoter in the absence of cotransfected PPARgamma. 15d-PGJ2 did not block nuclear translocation or DNA-binding activity of the transcription factor NFkappaB, but it did inhibit the activity of an NFkappaB reporter construct, suggesting that the mechanism of suppression of microglial iNOS by 15d-PGJ2 may involve interference with NFkappaB transcriptional activity in the nucleus. Thus, our data suggest the existence of a novel pathway mediated by cyclopentenone prostaglandins, which may represent part of a feedback mechanism leading to the cessation of inflammatory glial responses in the brain.

Molecular characterization of a gene for alanine aminotransferase from rice (Oryza sativa).

A cDNA clone encoding alanine aminotransferase (AlaAT) has isolated from randomly sequenced clones derived from a cDNA library of maturing rice seeds by comparison to previously identified genes. The deduced amino acid sequence was 88% and 91% homologous to those of the enzymes from barley and broomcorn millet (Panicum miliaceum), respectively. Using this cDNA as a probe, we isolated and sequenced the corresponding genomic clone. Comparison of the sequences of the cDNA and the genomic gene revealed that the coding region of the gene was interrupted by 14 introns 66 to 1547 bp long. Northern and western blotting analyses showed that the gene was expressed at high levels in developing seeds. When the 5'-flanking region between -930 and +85 from the site of initiation of transcription was fused to a reporter gene for beta-glucuronidase (GUS) and then introduced into the rice genome, histochemical staining revealed strong GUS activity in the inner endosperm tissue of developing seeds and weak activity in root tips. Similar tissue-specific expression was also detected by in situ hybridization. These results suggest that AlaAT is involved in nitrogen metabolism during the maturation of rice seed.