Requirement of alkanes for salt tolerance of Cyanobacteria: characterization of alkane synthesis genes from salt-sensitive Synechococcus elongatus PCC7942 and salt-tolerant Aphanothece halophytica.

Cyanobacteria have been attracting great interest in the research area of biofuel production. All Cyanobacteria contain C15 -C19 hydrocarbons, but physiological roles of hydrocarbons remain to be clarified. Recently, two universal but mutually exclusive hydrocarbon production pathways in Cyanobacteria were discovered. In this study, we constructed a deletion mutant of alkane synthesis genes in fresh water cyanobacterium Synechococcus elongates PCC 7942. The mutant was incapable to produce alkanes and exhibited normal growth phenotype at low salinity. But, the mutant became salt sensitive. Overexpression of alkane synthesis genes from halotolerant Aphanothece halophytica in Synechococcus PCC7942 restored the growth defect. The alkane synthesis gene from halotolerant cyanobacterium A. halophytica was salt induced and produced a significant amount of alkanes at high salinity. These results indicate the requirement of alkanes for salt tolerance, and the alkane synthesis genes from A. halophytica could be a promising candidate for future biofuel application. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyanobacteria have been attracting great interest in the research area of biofuel production. All Cyanobacteria contain C15 -C19 hydrocarbons, but physiological roles of hydrocarbons remain to be clarified. In this study, it was found that the deletion mutant of alkane synthesis genes in fresh water cyanobacterium Synechococcus elongates PCC 7942 was incapable to produce alkanes and salt sensitive. The alkane synthesis gene from halotolerant cyanobacterium Aphanothece halophytica was salt induced and produced a significant amount of alkanes at high salinity. These results demonstrate the alkane synthesis genes from A. halophytica could be a promising candidate for future biofuel application.

Layer-specific genes reveal a rudimentary laminar pattern in human nodular heterotopia.

OBJECTIVE: To define distinctive features of nodular heterotopia in specimens derived from drug-resistant patients with epilepsy by evaluating mRNA expression of three different layer-specific markers: Rorbeta, Er81, and Nurr1. METHODS: We analyzed the expression profile of these genes, recognized as markers mainly expressed in layer IV for Rorbeta, in layer V for Er81, and in layer VI for Nurr1, in surgical samples from 14 epileptic patients, using in situ hybridization. Six patients had subcortical nodular heterotopia and 8 patients were controls. The intrinsic organization of nodular formations and of the overlaying neocortex was assessed. RESULTS: In all patients, the 3 selected genes showed high cortical laminar specificity. In subcortical nodular heterotopia, the different gene expression profiles revealed a rudimentary laminar organization of the nodules. In the overlaying cortex, fewer cells expressed the 3 genes in the appropriate specific layer as compared to controls. CONCLUSIONS: These data provide new insights into possible ontogenetic mechanisms of nodular heterotopia formation and show the potential role of layer-specific markers to elucidate the neuropathology of malformations of cortical development.

Expression of layer-specific markers in the adult neocortex of BCNU-Treated rat, a model of cortical dysplasia.

The experimental model of cortical dysplasia (CD) obtained by administering carmustine (1-3-bis-chloroethyl-nitrosurea [BCNU]) in pregnant rat uterus mimics the histopathological abnormalities observed in human CD patients: altered cortical layering, and presence of heterotopia and dysmorphic/heterotopic neurons. To investigate further the cortical layering disruption and the neuronal composition of heterotopia in BCNU-exposed cortex, we analyzed the expression pattern of the transcription factors Nurr1, Er81, Ror-beta, and Cux2 (respectively specific markers of layers VI, V, IV and superficial layers) in the cortical areas of BCNU-treated rats by means of in situ hybridization, and compared the findings with those observed in adult control rats. Combining in situ hybridization and immunohistochemistry we also investigated the origin of dysmorphic or heterotopic neurons. The main results of the present study are (i) the analysis of cortical layer thickness revealed that the cortical thinning in the BCNU model was prevalently restricted to the superficial layers; (ii) in cortical and periventricular heterotopia, the prevalent presence of superficial layer neurons in the internal areas, and deeper layer neurons in a more peripheral region, demonstrated a rudimentary pattern of laminar organization in nodule formation; and (iii) the Er81 signal in the dysmorphic and heterotopic pyramidal neurons located in layers I/II showed that they belong to layer V. These results shed light on the disorganization of the laminar architecture of the BCNU model by providing correlations with normal cortical layering and revealing the ontogenesis of heterotopia and heterotopic/dysmorphic neurons. They also provide strong evidence of the usefulness of layer-specific markers in investigating the neuropathology of CD, thus opening up the possibility of expanding their application to human neuropathology.

Functional role of the secondary visual cortex in multisensory facilitation in rats.

Recent studies reveal that multisensory convergence can occur in early sensory cortical areas. However, the behavioral importance of the multisensory integration in such early cortical areas is unknown. Here, we used c-Fos immunohistochemistry to explore neuronal populations specifically activated during the facilitation of reaction time induced by the temporally congruent audiovisual stimuli in rats. Our newly developed analytical method for c-Fos mapping revealed a pronounced up-regulation of c-Fos expression particularly in layer 4 of the lateral secondary visual area (V2L). A local injection of a GABA A receptor agonist, muscimol, into V2L completely suppressed the audiovisual facilitation of reaction time without affecting responses to unimodal stimuli. Such a selective suppression was not found following the injection of muscimol into the primary auditory and visual areas. To examine whether or not the rats might have shown the facilitated responses because of increment of stimulus intensity caused by the two modal stimuli, the behavioral facilitation induced by the high-intensity unimodal stimuli was tested by the injection of muscimol into V2L, which turned out not to affect the facilitation. These results suggest that V2L, an early visual area, is critically involved in the multisensory facilitation of reaction time induced by the combination of auditory and visual stimuli.

Antagonism of p66shc by melanoma inhibitory activity.

The p66shc protein governs oxidant stress and mammalian lifespan. Here, we identify melanoma inhibitory activity (MIA), a protein secreted by melanoma cells, as a novel binding partner and antagonist of p66shc. The N-terminal collagen homology-2 (CH2) domain of p66shc binds to the Src Homology-3 (SH3)-like domain of MIA in vitro. In cells, ectopically expressed MIA and p66shc colocalize and co-precipitate. MIA also co-precipitates with the CH2 domain of p66shc in vivo. MIA expression in vivo suppresses p66shc-stimulated increase in endogenous hydrogen peroxide (H(2)O(2)), and inhibits basal and H(2)O(2)-induced phosphorylation of p66shc on serine 36 and H(2)O(2)-induced death. In human melanoma cells expressing MIA, endogenous MIA and p66shc co-precipitate. Downregulation of MIA in melanoma cells increases basal and ultraviolet radiation (UVR)-induced phosphorylation of p66shc on serine 36, augments endogenous H(2)O(2) levels, and increases their susceptibility to UVR-induced death. These findings show that MIA binds to p66shc, and suggest that this interaction antagonizes phosphorylation and function of p66shc.

Gene expression profiling of primate neocortex: molecular neuroanatomy of cortical areas.

One hundred years have passed since Brodmann's mapping of the mammalian neocortex. Solely on the basis of morphological observations, he envisaged the conservation and differentiation of cortical areal structures across various species. We now know that neurochemical, connectional and functional heterogeneity of the neocortex accompanies the morphological divergence observed in such cytoarchitectonic studies. Nevertheless, we are yet far from fully understanding the biological significance of this cortical heterogeneity. In this article, we review our past works on the gene expression profiling of the postnatal primate cortical areas, by quantitative real-time polymerase chain reaction (PCR), DNA array, differential display PCR and in situ hybridization analyses. These studies revealed both the overall homogeneity of gene expression across different cortical areas and the presence of a small number of genes that show markedly area-specific expression patterns. In situ hybridization showed that, among such genes, occ1 and retinol-binding protein (RBP) mRNAs are selectively expressed in the neuronal populations that seem to be involved in distinct neural processing such as sensory reception (for occ1) and associative function (for RBP). Such a molecular neuroanatomical approach has the promise to provide an important link between structure and function of the cerebral cortex.

7-12 Hz cortical oscillations: behavioral context and dynamics of prefrontal neuronal ensembles.

7-12 Hz Oscillations, characterized by spindle-like high-voltage rhythmic spike components, appear in quiet immobile states of rats. However, it remains unclear what their relationships with preceding behavioral activities are and how prefrontal neuronal dynamics during these oscillations is. In the present study, we first determined the relationship of 7-12 Hz oscillations with the wake-sleep cycle and preceding behavioral activities in several normal rat strains by recording electroencephalograms from the multiple cortical regions. Prolonged awake period transiently enhanced the following appearance of 7-12 Hz oscillations, which were frequently followed by slow-wave sleep. The degree of transient enhancement under the task condition was similar to that by prolonged wakefulness under the no-task condition. In addition, by recording local-field potential and multi-unit activities in the medial prefrontal cortex, we determined the temporal dynamics of prefrontal neuronal activities in relation to 7-12 Hz oscillations. Collective neuronal activities in medial prefrontal cortex were gradually organized into phase-locked patterns and showed highly synchronization during these oscillations. These dynamics were in temporal proximity to those of slow-wave activities (<4 Hz). Since slow-wave activities are thought to synchronize large spatial domains, these results suggest that 7-12 Hz oscillations are involved in the transition from the awake to sleep states by oscillatory entrainment of global cortical networks including the prefrontal neurons.

A voltage-gated potassium channel, Kv3.1b, is expressed by a subpopulation of large pyramidal neurons in layer 5 of the macaque monkey cortex.

In the cerebral cortex, the voltage-gated potassium channel, Kv3.1b, a splicing variant of Kv3.1, has been associated with fast-firing interneurons. Here, we report strong expression of Kv3.1b-protein and mRNA in both Betz and Meynert pyramidal cells of the monkey, as shown by immunohistochemistry and in situ hybridization. Strong expression also occurs in large pyramidal neurons in layer 5 of several cortical areas. In addition, most of these Betz and layer 5 pyramids, and about 10% of the labeled Meynert cells weakly co-expressed the calcium binding protein parvalbumin. Electron microscopy shows that the expression of Kv3.1b is localized to the somal and proximal dendritic cytoplasmic membrane, as expected for a channel protein. These results suggest that some large pyramidal neurons may constitute a functional subpopulation, with a distinctive distribution of voltage-gated potassium channels capable of influencing their repetitive firing properties.

Characterization of JNK-like protein derived from a mosquito cell line, C6/36.

When Western blot analysis of heat-killed bacteria- and lipopolysaccharide (LPS)-treated Aedes albopictus mosquito cell line C6/36 was performed using antiphospholyrated c-Jun amino-terminal kinase (JNK) antibodies, approximately 46 kDa protein was clearly detected with a peak around 30 min. After the C6/36 cells were incubated at 45 degrees C in order to induce apoptosis, the 46 kDa protein continued to be detected for at least 3 h. The internalization of fluorescein-labelled bacteria was inhibited by a JNK-specific inhibitor SP600125, suggesting that phagocytosis involves the JNK signalling pathway in mosquito cells. Based on these results, we found one candidate for the nucleotide sequence of JNK (Ae-JNK) from the C6/36 cells. This study is the first report regarding the mitogen-activated protein kinase (MAPK) of mosquito.

Synaptic long-term depression (LTD) in vivo recorded on the rat cerebellar cortex.

Long-Term Depression (LTD) of the parallel fiber synapses of the cerebellar cortex has been intensively studied over the last 20 years and is now considered to be a physiological mechanism underlying learning and memory of the cerebellar cortex. With microelectrode recording in vivo, the induced LTD is recorded reliably up to 2 hours. Using surface electrodes we have recorded parallel fiber responses due to the currents generated by the AMPA type receptors of the dendritic spines in the intact vermal cortex of decerebrated rats. We have found that by conjunctively stimulating the climbing and parallel fiber pathways, an LTD was induced which persisted for as long as the recording conditions permitted. The longest lasting LTD of our present results was for 5 hours.

Induction of long-term depression in cerebellar Purkinje cells requires a rapidly turned over protein.

Evidence is presented indicating that the induction of long-term depression (LTD) in Purkinje cells (PCs) requires a rapidly turned over protein(s) during a critical time period within 15 min after the onset of LTD-inducing stimulation and that synthesis of this protein is maintained by mRNAs supplied via transcription. LTD was induced in granule cell axon (GA)-to-PC synapses by stimulation of these synapses at 1 Hz for 5 min in conjunction with the climbing fibers (CFs) forming synapses on the same PCs and represented by a persistent reduction in the GA-induced excitatory postsynaptic potentials (EPSPs). Not only a prolonged but also a brief (5 min) pulse application of translational inhibitors (anisomycin, puromycin, or cycloheximide) effectively blocked the LTD induction. Pulses applied during the period from 30 min before to 10 min after the onset of conjunctive stimulation blocked the LTD induction, but those applied 15 min after were ineffective. The three translational inhibitors blocked the LTD induction similarly, suggesting that the effect is due to their common action of inhibiting protein synthesis. Infusion of a mRNA cap analogue (7-methyl GTP) into PCs also blocked LTD induction, ensuring that the postsynaptic protein synthesis within PCs is required for LTD induction. Transcriptional inhibitors, actinomycin D and 5,6-dichloro-l-beta-D-ribofuranosyl-benzimidazole, also blocked the LTD induction, but this effect was apparent when 5-min pulses of the transcriptional inhibitors preceded the conjunctive stimulation by 30 min or more. This time lag of 30 min is presumed to be required for depletion of the protein(s) required for LTD induction. The presently observed effects of translational and transcriptional inhibitors on the LTD induction are of temporal characteristics corresponding to their depressant effects on the type-1 metabotropic glutamate-receptor (mGluR1)-mediated slow EPSPs in PCs as we have reported recently. An antagonist of mGluR1s [(RS)-1-aminoindan-1,5-dicarboxylic acid], however, did not block LTD induction when it was applied during the 10-min period following conjunctive stimulation, where translational inhibitors effectively blocked LTD induction. This discrepancy in time course suggests that the rapidly turned over protein(s) required for LTD induction is involved in a process occurring downstream of the activation of mGluR1s.

Electroretinogram analysis of relative spectral sensitivity in genetically identified dichromatic macaques.

The retinas of macaque monkeys usually contain three types of photopigment, providing them with trichromatic color vision homologous to that of humans. However, we recently used molecular genetic analysis to identify several macaques with a dichromatic genotype. The affected X chromosome of these animals contains a hybrid gene of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) photopigments instead of separate genes encoding L and M photopigments. The product of the hybrid gene exhibits a spectral sensitivity close to that of M photopigment; consequently, male monkeys carrying the hybrid gene are genetic protanopes, effectively lacking L photopigment. In the present study, we assessed retinal expression of L photopigment in monkeys carrying the hybrid gene. The relative sensitivities to middle-wavelength (green) and long-wavelength (red) light were measured by electroretinogram flicker photometry. We found the sensitivity to red light to be extremely low in protanopic male monkeys compared with monkeys with the normal genotype. In female heterozygotes, sensitivity to red light was intermediate between the genetic protanopes and normal monkeys. Decreased sensitivity to long wavelengths was thus consistent with genetic loss of L photopigment.

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein.

It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.

Similarity and variation in gene expression among human cerebral cortical subregions revealed by DNA macroarrays: technical consideration of RNA expression profiling from postmortem samples.

The functional regionality of the human cerebral cortex suggests that a set of genes might be activated in each subregion of the neocortex to support its specific functions. To test this hypothesis, we employed the DNA array technique to compare the mRNA expression profiles of three neocortical subregions of the human brain: prefrontal cortex (Area 46), motor cortex (Area 4) and visual cortex (Area 17). The macroarray analysis on high quality mRNA from postmortem brains revealed that the expression profiles of the different cortical areas are almost similar: only six out of 1088 known genes exhibited significant differences (>2-fold) in their expression. RT-PCR studies with an increased number of samples confirmed that expression of only two genes, annexin II and early growth response protein 1, varied by 2-fold among the regions, whereas expression of the others showed large inter-individual difference. These results suggest that the whole neocortex of humans is more homogeneous than we expected at the level of gross gene expression profiles. In parallel, sensitivity and accuracy of radioisotope-based DNA macroarrays and fluorescence-based DNA microarrays were tested.

ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide.

We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O2*- generation during phagocytosis in human polymorphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zymosan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of Cu,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-adducts were present, cells after stimulation were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), the DEPMPO-OOH was detected in extracellular fluid but not in the cell fraction. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O2*- of extracellular space including the intraphagosome but not that of intracellular space in sOZ-stimulated phagocytes.

Growth/differentiation factor 7 is preferentially expressed in the primary motor area of the monkey neocortex.

We applied a differential display PCR technique to isolate molecules that are area-specific in expression in the primate neocortex, and found that growth/differentiation factor 7 (GDF7), a member of the bone morphogenetic protein (BMP)/transforming growth factor (TGF) beta super-family, is preferentially expressed in the primary motor area of African green monkeys (Cercopithecus aethiops). We proved that GDF7 is 10 times more abundant in the motor cortex than in the visual cortex by northern blotting and quantitative RT-PCR. When we examined the neocortex of closely related rhesus monkeys (Macaca mulatta), GDF7 was also most abundant in the motor cortex, although the regional difference was reduced to 3-fold. This differential expression pattern was observed in both newborn and infant rhesus monkeys. We found that several type I/II receptors of BMP, candidates of the receptors for GDF7, are uniformly expressed in the mature neocortex. The unique expression pattern of GDF7 suggests that it may play an active role in the motor area of the primate neocortex.

The occ1 gene is preferentially expressed in the primary visual cortex in an activity-dependent manner: a pattern of gene expression related to the cytoarchitectonic area in adult macaque neocortex.

Marker molecules to visualize specific subsets of neurons are useful for studying the functional organization of the neocortex. One approach to identify such molecular markers is to examine the differences in molecular properties among morphologically and physiologically distinct neuronal cell types. We used differential display to compare mRNA expression in the anatomically and functionally distinct areas of the adult macaque neocortex. We found that a gene, designated occ1, was preferentially transcribed in the posterior region of the neocortex, especially in area 17. Complete sequence analysis revealed that occ1 encodes a macaque homolog of a secretable protein, TSC-36/follistatin-related protein (FRP). In situ hybridization histochemistry confirmed the characteristic neocortical expression pattern of occ1 and showed that occ1 transcription is high in layers II, III, IVA and IVC of area 17. In addition, occ1 transcription was observed selectively in cells of the magnocellular layers in the lateral geniculate nucleus (LGN). Dual labeling immunohistochemistry showed that the occ1-positive neurons in area 17 include both gamma-aminobutyric acid (GABA)-positive aspiny inhibitory cells and the alpha-subunit of type II calcium/calmodulin-dependent protein kinase (CaMKII alpha)-positive spiny excitatory cells. With brief periods of monocular deprivation, the occ1 mRNA level decreased markedly in deprived ocular dominance columns of area 17. From this we conclude that the expression of occ1 mRNA is present in a subset of neurons that are preferentially localized in particular laminae of area 17 and consist of various morphological and physiological neuronal types, and, furthermore, occ1 transcription is subject to visually driven activity-dependent regulation.

[A case report of TS-1 in a patient with advanced gastric cancer].

TS-1 is an oral anticancer drug that produces biochemical modulation. TS-1 is composed of FT (tegafur), CDHP (gimestat, which inhibits 5-FU degradation enzyme), and Oxo (otastat potassium, which reduces 5-FU gastrointestinal toxicities), in a molar ratio of 1:0.4:1. We administered TS-1 to a 68-year-old female gastric cancer patient, after distal gastrectomy (Stage IV, cur C). As a result of abdominal CT, the diameter of metastatic lymph node increased before and after surgery, and before TS-1 (45 x 35 mm), but it was reduced after 1 course of TS-1 (37 x 25 mm), 2 courses of TS-1 (35 x 20 mm), 3 courses of TS-1 (30 x 20 mm), 4 courses of TS-1 (30 x 20 mm), and 6 months after 4 courses of TS-1 (20 x 20 mm). The reduction rate is 74.6%. The value of CA125 was reduced 74.4 to 8.6 after TS-1. Anorexia and back pain, which occurred after operation, disappeared after TS-1. There was no side effect over grade 3.

Rapidly turned over protein maintains metabotropic synaptic transmission in Purkinje cells.

It has generally been thought that protein synthesis is required for relatively slow cellular processes such as synaptogenesis and synaptic plasticity. In this study on rat cerebellar slices, we found that metabotropic glutamate receptor-mediated synaptic transmission to cerebellar Purkinje cells was quickly and persistently depressed by brief (5 min) applications of translational inhibitors, which were confirmed to induce quick and persistent depression of protein synthesis in cerebellar tissues. Brief applications of transcriptional inhibitors also depressed metabotropic synaptic transmission, but progressively over 1 h, presumably due to depletion of mRNAs in the dendrites. Results of this study indicate the presence of a unique protein(s) that is dynamically involved in metabotropic synaptic transmission.