Bacillus cereus nosocomial infection from reused towels in Japan.

It was noticed that there was an increase in Bacillus cereus nosocomial infections in the summer from 2000 to 2005. In 2005, five bloodstream infections occurred in five patients related to catheter use. The causative strains were distinct from each other and belonged to novel multilocus sequence types (ST): ST365, ST366, ST367 and ST368. Two ST365 strains from two patients were further distinguished by pulsed-field gel electrophoresis. B. cereus contamination was observed with reused (dried and steamed) towels (>10(6)cfu/towel) and washing machines in hospital linen rooms. B. cereus strains from towels belonged to ST167, ST365, ST380 and ST382, and a proportion of these were the same, or similar, to strains from patients. All the hospital strains of B. cereus were distinct from those from food-poisoning strains (ST26, ST142, ST381). Ciprofloxacin resistance was observed only in hospital strains. Neither emetic toxin nor cytotoxin K gene, usually present in food poisoning strains, were found in the hospital strains, except for one patient isolate. The data suggest that specific B. cereus strains are circulating within a hospital, with genotypes, antibiotic susceptibilities and virulence gene patterns generally distinct from those of food poisoning, and that in Japan, towels are an important source of contamination, especially in summer.

Macrolide-lincosamide-streptogramin B resistance phenotypes and genotypes among Staphylococcus aureus clinical isolates in Japan.

In total, 269 methicillin-resistant Staphylococcus aureus (MRSA) and 434 methicillin-susceptible S. aureus (MSSA) isolates were investigated to determine their macrolide-lincosamide-streptogramin B (MLS(B)) resistance phenotypes and genotypes. The constitutive phenotype (61.3% in MRSA, 1.3% in MSSA) and erm(A) gene predominated among the 261 erythromycin-resistant MRSA isolates, while the inducible phenotype (38.7% in MRSA, 94.0% in MSSA) and erm(C) gene were more prevalent among the 150 erythromycin-resistant MSSA isolates. There was a higher incidence of the MLS(B) inducible phenotype compared with other countries, perhaps because MLS(B) antibiotics are not recommended as first-line agents against S. aureus in Japan.

Progesterone inhibits apolipoprotein-mediated cellular lipid release: a putative mechanism for the decrease of high-density lipoprotein.

In order to investigate the mechanism for female gonadal hormones to regulate the plasma high-density lipoprotein (HDL) level, the effect of 17 beta-estradiol and progestogens was examined in vitro on the assembly of HDL by free apolipoprotein A-I (apoA-I) with cellular cholesterol and phospholipid. ApoA-I generated HDL particles by removing cholesterol and phospholipid from human fibroblasts, MRC-5. While 17 beta-estradiol did not influence this reaction, progesterone suppressed the removal by apoA-I of both cholesterol and phospholipid, with the extent of the inhibition more for cholesterol than phospholipid. Three other synthetic progestogens showed the similar inhibitory effect on the cellular cholesterol release. Cellular cholesterol de novo-synthesized from mevalonolactone entered more into the acyl-esterified cholesterol compartment and less to the unesterified compartment in the presence of progesterone. On the other hand, progesterone did not influence the overall mass ratio of free and esterified cholesterol in the cell. Cell-surface cholesterol was also uninfluenced by progesterone when probed by extracellular cholesterol oxidase reaction or by diffusion-mediated cellular cholesterol release to cyclodextrin. Neither caveolin-1 nor ABCA1 expression was influenced by progesterone. Progesterone thus seems primarily to alter the specific intracellular cholesterol compartment that is related to the apoA-I-mediated HDL assembly. This mechanism might contribute to the decrease of plasma HDL by administration of progestogen in women under hormone replacement therapy.

Human ABCA1 contains a large amino-terminal extracellular domain homologous to an epitope of Sjögren's Syndrome.

ABCA1 has been suggested to play a key role in cellular lipid release from peripheral cells. In order to study structure-function relationship of this protein, the protein product of a full-length human ABCA1 cDNA was examined for its functions and topological orientation. The electrophoretic mobilities of human ABCA1 expressed in transfected cells increased when treated with N-glycosidase F, suggesting that ABCA1 is highly glycosylated. The ABCA1 was photoaffinity-labeled with ATP and mediated the apoA-I-dependent-release of cholesterol and phospholipid. The influenza hemagglutinin (HA) epitope was introduced into the amino-terminus (N-HA) or between the residues 207 and 208 (207-HA) of the protein. While an antibody against the C-terminus peptide of ABCA1 detected both fusion proteins, an anti-HA antibody did not react with the N-HA fusion protein. Confocal microscopy demonstrated strong cell surface signal with the anti-HA antibody of nonpermeabilized HEK293 cells expressing the 207-HA fusion protein. The results suggested that the signal peptide in the amino-terminal region is cleaved off in its mature form and that the following large hydrophilic region is exposed to outside of cells unlike previously proposed models. We found that this amino-terminal extracellular domain contains a segment homologous to the autoantigen SS-N, an epitope of Sjögren's syndrome, and further identified that ABCA7 codes for the autoantigen SS-N.

Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells.

High density lipoprotein (HDL) is assembled by interaction of apolipoprotein A-I with human monocytic leukemia cell line THP-1 by removing cellular cholesterol and phospholipid. Although the HDL formed with undifferentiated THP-1 cells contained only phosphatidylcholine and almost no cholesterol, the cells differentiated with phorbol 12-myristate 13-acetate (PMA) generated HDL enriched in cholesterol. The extent of cholesterol enrichment related to the cellular cholesterol level in the differentiated cells, but only weakly in the undifferentiated cells. In contrast, the differentiation had no influence on the diffusion-mediated cellular cholesterol efflux. The undifferentiated cells expressed the messages of ATP-binding cassette transporter 1 and caveolin-1, at low levels, and the PMA-induced differentiation resulted in substantial expression of both messages. Caveolin-1 protein expression was also highly induced by the PMA treatment of THP-1 cells. When the cells were treated with the antisense DNA of caveolin-1 and differentiated, both caveolin-1 synthesis and cholesterol incorporation into the HDL were reduced in parallel to generate the cholesterol-poor HDL. We concluded that caveolin-1 is involved in enrichment with cholesterol of the HDL generated by the apolipoprotein-cell interaction. This function is independent of the assembly of HDL particles with cellular phospholipid and of nonspecific, diffusion-mediated efflux of cellular cholesterol.

Enhancement of the cAMP-induced apolipoprotein-mediated cellular lipid release by calmodulin inhibitors W7 and W5 from RAW 264 mouse macrophage cell line cells.

Apolipoprotein (apo) A-I generates high-density lipoprotein (HDL) by removing cellular cholesterol and phospholipid on the interaction with cells as a main source of plasma HDL. The reaction is induced by dibutylyl cyclic (dbc) adenosine monophosphate (AMP) in RAW 264, mouse macrophage cell line cells, and we investigated its pharmacologic modulation using this cell model. Release of cellular cholesterol and choline phospholipid by apoA-I was increased 9.9 and 4.2 times, respectively, by pretreatment of the cells with 300 microM dbcAMP for 24 h. Calmodulin inhibitors, W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) and W5 (N(6-aminohexyl)-1-naphthalenesulfonamide), increased the apoA-I-mediated lipid release by 3 times from the dbcAMP-treated cells. The optimal drug concentrations (80 and 160 microM for W7 and W5, respectively) were not parallel with those reported for in vitro calmodulin inhibition (IC50, 28 and 240 microM for W7 and W5, respectively, toward phosphodiesterase activity), and in fact 40 microM W7 showed much stronger intracellular calmodulin inhibition than did 300 microM W5 using S7AAS2, a fluorescent peptide probe. Other calmodulin inhibitors such as amitriptyline, chlorpromazine, and trifluoperazine showed no effect on the apoA-I-mediated cholesterol release. In contrast to these results, neither dbcAMP nor W7 influenced the diffusion-mediated nonspecific cholesterol efflux to lipid microemulsion. We concluded that W7 and W5 increased the interaction of apoA-I with RAW 264 cells to generate more HDL. The effect did not seem directly correlated to their cal modulin inhibition or modulation of cAMP and protein kinase C.

Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.

Murine macrophage RAW264 were investigated for their response to lipid-free apolipoproteins. Preincubation of the cells with 300 microM dibutyryl cyclic (dBc) AMP for 16 h induced specific binding of apolipoprotein (apo) A-I to the cells and apoA-I-mediated HDL formation with cellular lipids, neither of which was detected in the absence of dBcAMP. Dose-dependent changes of the apoA-I specific binding and the apoA-I-mediated cholesterol release were largely superimposable. ApoA-II also mediated lipid release after the treatment of the cells with dBcAMP and effectively displaced the apoA-I binding to the cells. In contrast, cellular cholesterol efflux to lipid microemulsion and to 2-(hydroxypropyl)-beta-cyclodextrin was uninfluenced by the dBcAMP treatment. To induce the cellular reactivity with apoA-I, the incubation with dBcAMP required at least 6 h. Actinomycin D, cycloheximide, puromycin, and brefeldin A suppressed both the induction of apoA-I-mediated lipid release and the apoA-I specific binding to the cells. Analysis of the expression level of ABC1 mRNA by using reverse transcription-polymerase chain reaction and oligonucleotide arrays revealed that ABC1 mRNA was already expressed in the dBcAMP-untreated cells, and the dBcAMP treatment for 16 h enhanced its expression 9-13-fold. We conclude that dBcAMP selectively induces apolipoprotein-mediated cellular lipid release and accordingly high-density lipoprotein generation by inducing specific binding of apolipoprotein, but does not influence diffusion-mediated lipid efflux. The cell-apolipoprotein interaction seems to depend on cellular protein biosynthesis and transport. A substantial increase in the level of ABC1 mRNA caused by the dBcAMP treatment indicates that ATP-binding cassette transporter 1, the protein product of ABC1, may directly be responsible for the interaction, but the question about the absence of the interaction with its baseline expression level remains.

Identification of cis-acting elements in the proximal promoter region for brain-specific exon 1 of the mouse aromatase gene.

Among multiple exons 1 of the mouse aromatase gene, brain-specific exon 1 is only utilized in the hypothalamus and amygdala regions. In this study, identification of the promoter region necessary for basal transcription of the aromatase gene in the brain was undertaken. Deletions of various lengths were introduced into the overall promoter region, which was fused to the chloramphenicol acetyltransferase gene. The resulting reporters were transfected into cultured neurons from the diencephala of fetal mouse brains on embryonic day 13 and then their CAT mRNA levels were determined. The reporter plasmid containing the promoter region 202 bp upstream from the transcriptional initiation site gave the greatest expression. Then binding of trans-acting factors in a nuclear extract of the diencephala to the -202 bp promoter region was investigated by DNase I footprint analysis, multiple protected areas, referred to as Arom-Aalpha, Abeta, Agamma, B and C, being found. Gel shift assays, performed with oligonucleotides corresponding to the protected areas, showed that nuclear DNA binding factors form specific complexes exhibiting different mobilities. Substitution in the Arom-Aalpha or -B sequence in the promoter region in the CAT reporters decreased the CAT mRNA expression levels to about one-fifth the wild type one. These results suggest that multiple nuclear factors bound to the core promoter region participate in the expression of the aromatase gene in mouse brain neurons.

Autonomous expression of aromatase during development of mouse brain is modulated by neurotransmitters.

A transient increase in aromatase activity is known to occur in the hypothalamus of rodents in pre- and postnatal periods. The mechanisms regulating such a developmental increase of brain aromatase was studied in fetal mouse diencephalic cells, by measuring aromatase mRNA levels by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. When slices of diencephalon were cultured on embryonic day (E) 12, E13 and E15, the level of aromatase mRNA continued to increase for the first 2 to 3 days. A time-dependent increase of mRNA was also shown for 3 days in E13 neuronal cells dissociated with papain and cultured in chemically defined medium. However, no significant increase was observed in E10 or E11 brain cells cultured by either method. Aromatase mRNA was detected in neither cerebral cortex neurons nor astrocytes. An alpha1-selective adrenergic agonist, phenylephrine, increased aromatase in the E13 diencephalic neurons in culture, whereas prazosin, an alpha1-antagonist, suppressed the mRNA level. Ligands for alpha2- or beta-adrenergic receptors did not alter the mRNA level. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide as well as phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP all increased the mRNA level. We concluded that: (a) the developmental increase of aromatase mRNA in diencephalic neurons is an autonomous event and is perhaps genetically regulated after E12; (b) aromatase mRNA is expressed in a cell type- and region-specific manner; and (c) protein kinases C and G activated via receptors of the specific neurotransmitters may be involved in modulation of the developmental expression of aromatase mRNA.

Neurotransmitter-mediated regulation of brain aromatase: protein kinase C- and G-dependent induction.

Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by alpha 1-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the alpha 1-agonist phenylephrine than in control neurons, whereas prazosin, an alpha 1-antagonist, suppressed this increase, and ligands for alpha 2- or beta-adrenergic receptors did not exert any influence. The profile of alpha 1-adrenergic receptor subtypes during actual development in vivo suggested that the alpha 1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyrylcyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via alpha 1 (possibly alpha 1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. beta-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.

In vitro increase of aromatase mRNA in diencephalic neurons.

This study was designed to (1) examine the ability of fetal diencephalic neurons cultured in vitro to express aromatase mRNA and (2) evaluate the involvement of several environmental factors which may regulate the development and differentiation of the neurons in the central nervous system. Brain cells from fetal mice at various developmental stages were cultured as tissue slices and as primary monolayer cells, and the expression levels of aromatase mRNA in the cultured cells were measured by a quantitative reverse transcription-polymerase chain reaction method using an internal standard. On cultured slices of the diencephalic region from fetal mice on embryonic day 12 (E12), E13, or E15 on collagen-coated membranes, the expression level of the mRNA continued to increase for the initial 2-3 days as that in vivo. Time-dependent increase of aromatase mRNA was also observed for 3 days in E13 neuronal cells dissociated with papain and cultured on poly L-Lys-coated dishes in serum-free medium. However, no significant time-dependent increase of the aromatase mRNA level was observed in E10 or E11 brain cells cultured by either method. These findings suggest that the developmental increase of aromatase mRNA in diencephalic neurons is an endogenous characteristic and probably genetically determined after E12.

Pre- and post-translational regulation of aromatase by steroidal and non-steroidal aromatase inhibitors.

Treatment of castrated quail with testosterone (T) reliably activates male copulatory behavior and, at the same time, increases the aromatase activity (AA), the number of aromatase-immunoreactive (ARO-ir) cells and the concentration of aromatase mRNA as measured by RT-PCR in the brain. All these effects can be mimicked by estrogens. The behavioral effects of T can be blocked by a variety of aromatase inhibitors and, in parallel, the AA is strongly inhibited in the preoptic area (POA). We showed recently that the steroidal inhibitor, 4-OH-androstenedione (OHA) markedly decreases the immunostaining density of brain ARO-ir cells while the non-steroidal inhibitor, R76713 (racemic Vorozole; VOR) unexpectedly increased the density of this staining, despite the fact that the enzyme activity was completely inhibited. To generalize these findings and try to identify the underlying mechanism, we compared here the effects of two steroidal (OHA and androstatrienedione [ATD]) and two non-steroidal (VOR and Fadrozole [FAD]) aromatase inhibitors on the aromatase immunostaining and aromatase mRNA concentration in the brain of castrated quail concurrently treated with T. The 4 inhibitors significantly blocked the activation by T of male copulation. The two steroidal inhibitors decreased the immunostaining of brain ARO-ir cells but both VOR and FAD markedly enhanced the density of this staining. In parallel, OHA and ATD completely blocked the T-induced increase in aromatase mRNA concentration, while VOR and FAD had no effect on these RNA concentrations in the POA-anterior hypothalamus and they decreased them only slightly in the posterior hypothalamus. Taken together these results suggest that the inhibition of AA by ATD or OHA and the subsequent removal of locally produced estrogens blocks the synthesis of aromatase presumably at the transcriptional level. By contrast, the two non-steroidal inhibitors tested here block AA but in parallel increase the aromatase immunostaining. This effect does not result from an enhanced transcription and it is therefore speculated that these compounds increase either the translation of the aromatase mRNA or the half-life of the protein itself.

Cell type- and region-specific expression of aromatase mRNA in cultured brain cells.

The expression of aromatase mRNA in cultured mouse brain cells was measured by a quantitative reverse transcription-PCR method using an internal standard. Aromatase mRNA was expressed in the cultured neurons prepared from diencephalon at 0.037 +/- 0.005 attomol/microgram total RNA. However, the mRNA was detected in neither the neurons from cerebral cortex nor astrocytes. These results demonstrate that expression of aromatase mRNA is regulated in cell type- and region-specific manners in cultured brain cells. The aromatase mRNA levels in neurons obtained from diencephalon were not affected by administration of testosterone, estradiol, dexamethasone, forskolin, or 12-O-tetradecanoyl 13-acetate. The results are in apparent disagreement with previous reports concerning regulation by androgens of brain aromatase activity in vivo and may suggest that aromatase expression in brain neurons is not directly induced by androgens. Androgen induction of brain aromatase may be mediated by several steps including cell-cell (neuron-neuron and/or neuron-glia) interaction.

Synergism between androgens and estrogens in the induction of aromatase and its messenger RNA in the brain.

It is established that testosterone (T) increases aromatase activity (AA) in the quail brain and that this induction of AA represents a limiting factor in the activation of male copulatory behavior. This action of T presumably results from an induction of aromatase synthesis since the number of aromatase-immunoreactive (ARO-ir) cells increases and, in parallel, there is an increase in aromatase mRNA as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) technology. The specific role of androgenic and estrogenic metabolites of T in this induction is not yet clear but product-formation assays suggest that both types of compounds synergize to increase AA. The exact role of androgens and estrogens in the induction of aromatase was examined by studying both the aromatase protein by immunocytochemistry and the aromatase mRNA by RT-PCR in castrated quail that had been treated with T or its androgenic metabolite, 5 alpha-dihydrotestosterone (DHT) or its estrogenic metabolite, estradiol-17 beta (E2) or both DHT and E2 simultaneously. A specific quantitative PCR technique using a modified aromatase as internal standard was developed for this purpose. T increased the number of ARO-ir cells in all brain areas and increased the concentration of ARO mRNA in the preoptic area-anterior hypothalamus (POA-aHYP) and in the posterior hypothalamus (pHYP). E2-treated birds had more ARO-ir cells than castrates in the posterior part of the medial preoptic nucleus (POM), in the bed nucleus stria terminalis (BNST) and tuber. Their aromatase mRNA concentration was significantly increased in the POA-aHYP but this effect did not reach significance in the pHYP. DHT by itself had no effect on either the number of ARO-ir cells (all brain regions considered) or the concentration of aromatase mRNA. DHT, however, synergized with E2, both in inducing ARO-ir neurons and in increasing aromatase mRNA concentration. This synergism was shown to be statistically significant in several brain areas. These data demonstrate that both androgens and estrogens regulate aromatase at the pretranslational level. Because the percentage increase in the number of ARO-iR cells was in general very similar to the increase in aromatase mRNA concentration, these data also suggest that these steroids regulate aromatase mostly by changing its mRNA synthesis or catabolism.

Bcl-2 gene is highly expressed during neurogenesis in the central nervous system.

An analytical method for quantitation of the RNA transcripts of murine bcl-2 gene was developed. The PCR products from bcl-2 alpha and bcl-2 beta mRNA were fluorometrically analyzed and their specific contents were calculated by the internal standard method. Both bcl-2 mRNAs in adult mice were transcribed at the highest level in the thymus and at a comparable level in the spleen. Aside from the immune system, the brain gave the most abundant levels of the bcl-2 mRNAs. The ratios of bcl-2 beta mRNA to bcl-2 alpha mRNA in the thymus and spleen were significantly higher than those in other tissues. During development of the brain, the bcl-2 alpha and bcl-2 beta mRNA levels were highest on embryonic day 15, and about two and three times higher than those of adult, respectively. The results suggest that the bcl-2 gene functions to regulate development and survival of neurons in the central nervous system.

Ca(2+)-binding proteins in rat synaptic fractions surveyed by the 45Ca2+ overlay method.

Ca(2+)-binding proteins in the synaptic and subsynaptic fractions (P2, synaptosome, synaptic plasma membrane, and postsynaptic density [PSD]-enriched fractions) and soluble fraction of rat brain were surveyed by a 45Ca2+ overlay method. The PSD-enriched fraction from cerebral cortex contained two major Ca(2+)-binding proteins (55,000 M(r) and 19,000 M(r)) and a distinct group (in 140,000 M(r) region), and two minor ones (66,000 M(r) and 16,000 M(r)); and the fraction from cerebellum contained two (55,000 M(r) and 19,000 M(r)). The proteins with 55,000 M(r) and 19,000 M(r) were identified as tubulin and calmodulin, respectively, and present in all the fractions investigated. The Ca(2+)-binding proteins of 140,000 M(r) region were found only in the PSD-enriched fraction isolated from cerebral cortex: neither the PSD-enriched fraction isolated from cerebellum nor other subcellular fractions prepared from cerebral cortex and cerebellum contained the proteins. The 140,000 M(r) Ca(2+)-binding proteins were the substrates for the Ca2+/calmodulin-dependent protein kinase II associated with PSD, and no change in the Ca(2+)-binding was detected by the 45Ca2+ overlay method after phosphorylation of the proteins by the protein kinase. The 16,000 M(r) Ca(2+)-binding protein might be the beta-subunit of calcineurin. Calretinin and calbindin-D28k were also detected as Ca(2+)-binding proteins in the soluble fractions of both cerebral cortex and cerebellum.

The presence of 17K Mr protein, a major specific substrate for kinase C, found in the triton-insoluble fraction of synaptosome prepared from rat brain.

Cytoskeletal preparation obtained from synaptosome fractions of rat cerebrum contained the activity of kinase C, which phosphorylated 17K Mr protein endogenous to the preparation. The kinase C activity associated with the synaptosome cytoskeletons is greater in the cerebellum and hippocampus than in the cerebrum. The enhancement rates of phosphorylation of the 17K Mr protein were 293%, 544%, and 526% in the Triton X-100-insoluble fractions of synaptosomes prepared from cerebral cortex, hippocampus, and cerebellum, respectively. The 17K Mr protein was distinct from myelin basic protein (MBP) for the following reasons: 1) The electrophoretic mobility of the protein was slightly smaller than that of major MBP of rat in the polyacrylamide gel of 10-20% linear gradient, and the protein was not contained in the purified rat myelin. 2) The isoelectric point of the protein was in neutral range, whereas that of MBP was in alkaline one. 3) The 17K Mr protein did not cross-react with anti-MBP antibody. The protein was shown to be a major substrate contained in the cytoskeletal preparation of synaptosome obtained from cerebrum except for contaminating MBP. Only serine residue of the 17K Mr protein was phosphorylated by the kinase C endogenous to the preparation. The results suggest strongly that the synaptic role of protein kinase C through phosphorylation of the 17K Mr protein.

P400 protein is one of the major substrates for Ca2+/calmodulin-dependent protein kinase II in the postsynaptic density-enriched fraction isolated from rat cerebral cortex, hippocampus and cerebellum.

Concanavalin A-binding glycoprotein with 250 K M(r) found in the postsynaptic density (PSD)-enriched preparation (or synaptic cytoskeleton) from rat cerebellum was identified with P400 protein from the physicochemical properties and enrichment in the cerebellum. Proteins homologous to the cerebellar 250 K M(r) protein occurred in the PSD-enriched preparations from rat cerebral cortex and from hippocampus, although the contents in the preparations were very low. The 250 K M(r) proteins in the PSD-enriched preparations from cerebellum and from cerebrum were highly phosphorylated by Ca2+/calmodulin (CaM)-dependent protein kinase II. The protein of synaptic plasma membrane (SPM) and PSD-enriched fractions prepared from cerebral cortex were not phosphorylated by the cAMP-dependent protein kinase endogenous to the fractions, whereas the protein from cerebellum was done in SPM and PSD-enriched fractions. The facts suggest that P400 or P400-like protein is closely associated with Ca2+/CaM-dependent protein kinase II in the PSD-enriched preparations, especially in the preparation from cerebral cortex. Phosphorylation of the protein by Ca2+/CaM-dependent protein kinase II may play an important role in the postsynaptic function in both cerebellum and at least in some areas of cerebrum.

Structural characterization of the human estrogen synthetase (aromatase) gene.

The estrogen synthetase (aromatase, cytochrome P-450AROM) gene has been isolated from human genomic libraries and characterized. The restriction map of 43 positive clones obtained indicated that this enzyme is present as a single copy gene. The aromatase gene is unexpectedly large compared with other forms of the cytochrome P-450 superfamily, spanning at least 70 kilobases. The gene consists of 10 exons and its 5'-untranslated region is divided into 2 exons by an intron of more than 35 kilobases long. This organization of the first exon in the aromatase gene is unique in the cytochrome P-450 superfamily. All the exon-intron junctional sequences conform to the canonical GT/AG rule. The sequences of a TATA box and a CAAT box are present 27 and 83 base pairs upstream from the transcriptional initiation site. Within 3 kilobases upstream from the initiation site, there are no typical consensus sequences of responsive elements for glucocorticoid and c-AMP, which regulate aromatase expression.