Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia.

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Expression patterns of SLIT/ROBO mRNAs reveal a characteristic feature in the entorhinal-hippocampal area of macaque monkeys.

OBJECTIVE: SLITs are secreted glycoproteins that bind to Roundabouts (ROBOs) which are a family member of transmembrane receptors. SLIT signaling has well-conserved roles in mediating axon repulsion in a developing nervous system. We previously reported that SLIT1 mRNA is enriched in middle layers of the prefrontal cortex of macaque monkeys in a developmentally regulated manner. Other SLIT (SLIT2 and SLIT3) mRNAs showed preferential expressions in the prefrontal cortex with a distinct laminar pattern. To obtain further clues to the role of SLIT signaling in the organization of the primate brain, we performed ISH analysis of SLIT and ROBO mRNAs using adult macaque brain tissues. RESULTS: In this study, we examined the expression patterns of SLITs and ROBOs (ROBO1 and ROBO2) in other brain regions, and found intense and characteristic expression patterns of these genes in the entorhinal-hippocampal area. In situ hybridization analysis revealed that SLIT1 and SLIT2 mRNAs showed marked complementary distribution in the entorhinal cortex. SLIT and ROBO mRNAs were widely expressed in the hippocampus with modest regional preference. These findings suggest that each SLIT gene has a specialized role that is particularly important for prefrontal as well as hippocampal connectivity in the primate cortex.

Properties of Zip4 accumulation during zinc deficiency and its usefulness to evaluate zinc status: a study of the effects of zinc deficiency during lactation.

Systemic and cellular zinc homeostasis is elaborately controlled by ZIP and ZnT zinc transporters. Therefore, detailed characterization of their expression properties is of importance. Of these transporter proteins, Zip4 functions as the primarily important transporter to control systemic zinc homeostasis because of its indispensable function of zinc absorption in the small intestine. In this study, we closely investigated Zip4 protein accumulation in the rat small intestine in response to zinc status using an anti-Zip4 monoclonal antibody that we generated and contrasted this with the zinc-responsive activity of the membrane-bound alkaline phosphatase (ALP). We found that Zip4 accumulation is more rapid in response to zinc deficiency than previously thought. Accumulation increased in the jejunum as early as 1 day following a zinc-deficient diet. In the small intestine, Zip4 protein expression was higher in the jejunum than in the duodenum and was accompanied by reduction of ALP activity, suggesting that the jejunum can become zinc deficient more easily. Furthermore, by monitoring Zip4 accumulation levels and ALP activity in the duodenum and jejunum, we reasserted that zinc deficiency during lactation may transiently alter plasma glucose levels in the offspring in a sex-specific manner, without affecting homeostatic control of zinc metabolism. This confirms that zinc nutrition during lactation is extremely important for the health of the offspring. These results reveal that rapid Zip4 accumulation provides a significant conceptual advance in understanding the molecular basis of systemic zinc homeostatic control, and that properties of Zip4 protein accumulation are useful to evaluate zinc status closely.

Diversity of retinal ganglion cells identified by transient GFP transfection in organotypic tissue culture of adult marmoset monkey retina.

The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs) besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC) have not yet been identified. Increasing interest has been shown in the common marmoset (Callithrix jacchus) monkey as a "super-model" of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells (midget, parasol, sparse, recursive, thorny, and broad thorny ganglion cells), and bistratified ganglion cells (recursive, large, and small bistratified ganglion cells [blue-ON/yellow-OFF-like]). By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina.

Fluorescent in situ hybridization technique for cell type identification and characterization in the central nervous system.

Central nervous system consists of a myriad of cell types. In particular, many subtypes of neuronal cells, which are interconnected with each other, form the basis of functional circuits. With the advent of genomic era, there have been systematic efforts to map gene expression profiles by in situ hybridization (ISH) and enhancer-trapping strategy. To make full use of such information, it is important to correlate "cell types" to gene expression. Toward this end, we have developed highly sensitive method of fluorescent dual-probe ISH, which is essential to distinguish two cell types expressing distinct marker genes. Importantly, we were able to combine ISH with retrograde tracing and antibody staining including BrdU staining that enables birthdating. These techniques should prove useful in identifying and characterizing the cell types of the neural tissues. In this article, we describe the methodology of these techniques, taking examples from our analyses of the mammalian cerebral cortex.

Prefrontal-enriched SLIT1 expression in Old World monkey cortex established during the postnatal development.

To elucidate the molecular basis of the specialization of cortical architectures, we searched for genes differentially expressed among neocortical areas of Old World monkeys by restriction landmark cDNA scanning . We found that mRNA of SLIT1, an axon guidance molecule, was enriched in the prefrontal cortex but with developmentally related changes. In situ hybridization analysis revealed that SLIT1 mRNA was mainly distributed in the middle layers of most cortical areas, robustly in the prefrontal cortex and faintly in primary sensory areas. The lowest expression was in the primary visual area. Analyses of other SLIT (SLIT2 and SLIT3) mRNAs showed preferential expression in the prefrontal cortex with a distinct laminar pattern. By contrast, the receptor Roundabout (ROBO1 and ROBO2) mRNAs were widely distributed throughout the cortex. Perinatally, SLIT1 mRNA was abundantly expressed in the cortex with modest area specificity. Downregulation of expression initially occurred in early sensory areas around postnatal day 60 and followed in the association areas. The prefrontal area-enriched SLIT1 mRNA expression results from a relatively greater attenuation of this expression in the other areas. These results suggest that its role is altered postnatally and that this is particularly important for prefrontal connectivity in the Old World monkey cortex.

Paraneoplastic antigen-like 5 gene (PNMA5) is preferentially expressed in the association areas in a primate specific manner.

To understand the relationship between the structure and function of primate neocortical areas at a molecular level, we have been screening for genes differentially expressed across macaque neocortical areas by restriction landmark cDNA scanning (RLCS). Here, we report enriched expression of the paraneoplastic antigen-like 5 gene (PNMA5) in association areas but not in primary sensory areas, with the lowest expression level in primary visual cortex. In situ hybridization in the primary sensory areas revealed PNMA5 mRNA expression restricted to layer II. Along the ventral visual pathway, the expression gradually increased in the excitatory neurons from the primary to higher visual areas. This differential expression pattern was very similar to that of retinol-binding protein (RBP) mRNA, another association-area-enriched gene that we reported previously. Additional expression analysis for comparison of other genes in the PNMA gene family, PNMA1, PNMA2, PNMA3, and MOAP1 (PNMA4), showed that they were widely expressed across areas and layers but without the differentiated pattern of PNMA5. In mouse brains, PNMA1 was only faintly expressed and PNMA5 was not detected. Sequence analysis showed divergence of PNMA5 sequences among mammals. These findings suggest that PNMA5 acquired a certain specialized role in the association areas of the neocortex during primate evolution.

Enriched expression of serotonin 1B and 2A receptor genes in macaque visual cortex and their bidirectional modulatory effects on neuronal responses.

To study the molecular mechanism how cortical areas are specialized in adult primates, we searched for area-specific genes in macaque monkeys and found striking enrichment of serotonin (5-hydroxytryptamine, 5-HT) 1B receptor mRNA, and to a lesser extent, of 5-HT2A receptor mRNA, in the primary visual area (V1). In situ hybridization analyses revealed that both mRNA species were highly concentrated in the geniculorecipient layers IVA and IVC, where they were coexpressed in the same neurons. Monocular inactivation by tetrodotoxin injection resulted in a strong and rapid (<3 h) downregulation of these mRNAs, suggesting the retinal activity dependency of their expression. Consistent with the high expression level in V1, clear modulatory effects of 5-HT1B and 5-HT2A receptor agonists on the responses of V1 neurons were observed in in vivo electrophysiological experiments. The modulatory effect of the 5-HT1B agonist was dependent on the firing rate of the recorded neurons: The effect tended to be facilitative for neurons with a high firing rate, and suppressive for those with a low firing rate. The 5-HT2A agonist showed opposite effects. These results suggest that this serotonergic system controls the visual response in V1 for optimization of information processing toward the incoming visual inputs.

Differential expression patterns of occ1-related genes in adult monkey visual cortex.

We have previously revealed that occ1 is preferentially expressed in the primary visual area (V1) of the monkey neocortex. In our attempt to identify more area-selective genes in the macaque neocortex, we found that testican-1, an occ1-related gene, and its family members also exhibit characteristic expression patterns along the visual pathway. The expression levels of testican-1 and testican-2 mRNAs as well as that of occ1 mRNA start of high in V1, progressively decrease along the ventral visual pathway, and end of low in the temporal areas. Complementary to them, the neuronal expression of SPARC mRNA is abundant in the association areas and scarce in V1. Whereas occ1, testican-1, and testican-2 mRNAs are preferentially distributed in thalamorecipient layers including "blobs," SPARC mRNA expression avoids these layers. Neither SC1 nor testican-3 mRNA expression is selective to particular areas, but SC1 mRNA is abundantly observed in blobs. The expressions of occ1, testican-1, testican-2, and SC1 mRNA were downregulated after monocular tetrodotoxin injection. These results resonate with previous works on chemical and functional gradients along the primate occipitotemporal visual pathway and raise the possibility that these gradients and functional architecture may be related to the visual activity-dependent expression of these extracellular matrix glycoproteins.

Comparative analysis of layer-specific genes in Mammalian neocortex.

We examined the expression patterns of 4 layer-specific genes in monkey and mouse cortices by fluorescence double in situ hybridization. Based on their coexpression profiles, we were able to distinguish several subpopulations of deep layer neurons. One group was characterized by the expression of ER81 and the lack of Nurr1 mRNAs and mainly localized to layer 5. In monkeys, this neuronal group was further subdivided by 5-HT2C receptor mRNA expression. The 5-HT2C(+)/ER81(+) neurons were located in layer 5B in most cortical areas, but they intruded layer 6 in the primary visual area (V1). Another group of neurons, in monkey layer 6, was characterized by Nurr1 mRNA expression and was further subdivided as Nurr1(+)/connective tissue growth factor (CTGF)(-) and Nurr1(+)/CTGF(+) neurons in layers 6A and 6B, respectively. The Nurr1(+)/CTGF(+) neurons coexpressed ER81 mRNA in monkeys but not in mice. On the basis of tracer injections in 3 monkeys, we found that the Nurr1(+) neurons in layer 6A send some corticocortical, but not corticopulvinar, projections. Although the Nurr1(+)/CTGF(-) neurons were restricted to lateral regions in the mouse cortex, they were present throughout the monkey cortex. Thus, an architectonic heterogeneity across areas and species was revealed for the neuronal subpopulations with distinct gene expression profiles.

Activity-dependent expression of occ1 in excitatory neurons is a characteristic feature of the primate visual cortex.

occ1 is a gene whose expression is particularly abundant in neurons in the macaque primary visual cortex (V1). In the present study, we report that the expression of occ1 mRNA in the macaque neocortex can be classified into two modes. The first mode is associated with excitatory neurons distributed in the major thalamocortical recipient layers that exhibit strong cytochrome oxidase activity. This is highly prominent in V1. The second mode is associated with parvalbumin-positive GABAergic interneurons and is distributed across the macaque neocortex. In V1, monocular deprivation showed that occ1 mRNA expression in excitatory neurons was markedly dependent on afferent activity, whereas that in GABAergic interneurons was not. Cross-species comparison showed specific differences in expression. In marmosets, a strong expression was observed in V1 similarly to macaques. The occ1 mRNA expression, however, was generally weak in the mouse neocortex. In rabbit and ferret cortices, the strong expression was observed only in GABAergic interneurons. We conclude that activity-dependent occ1 mRNA expression in the excitatory neurons of V1 was caused by a novel mechanism acquired by primates after their separation from other lineages.

Local design principles of mammalian cortical networks.

To understand global and local design principles of mammalian cerebral cortical networks, we applied network-theoretical approaches to connectivity data from macaque and cat cortical networks. We first confirmed "small-world" properties and searched for the evidence of hierarchical modularity. To elucidate their local design principles, we then compared these cortical networks, based on the significance profile (SP) of network motifs in the real network compared to randomized networks. We found that SPs of different mammalian cortical networks are highly conserved and robust, suggesting constraints of neocortical development and evolution.

Retinol-binding protein gene is highly expressed in higher-order association areas of the primate neocortex.

The neocortex consists of histochemically, connectionally, and functionally distinguishable areas. Recently, molecular biological techniques have enabled us to find rare types of genes expressed in specific neocortical areas. We previously reported occ1 gene as preferentially expressed in the primary visual cortex (V1), using the differential display method. Here, by differential display, we found selective and strong expression of the serum retinol-binding protein (RBP) gene, in higher-order association areas. In V1, RBP mRNA was expressed only in the superficial part of layer II, but its expression increased, involving deeper layers, along the visual pathway. In visual association areas such as TE, RBP mRNA was strongly expressed in both supra- and infragranular layers. In primary auditory and somatosensory areas, as in V1, RBP expression was low, and restricted to the upper part of the supragranular layers. The laminar pattern of RBP expression is in marked contrast with that of occ1; and in early visual areas where both genes are expressed, these occur in distinct sublayers within the supragranular layers. In neonatal monkeys, the area-specific expression pattern of RBP was less distinct, suggesting that the characteristic expression of RBP in higher-order association areas is mainly established postnatally.