Sudden infant death due to Lactococcal infective endocarditis.

Infective endocarditis (IE) of infants is rare, most of which occur associated with congenital heart disease or its cardiac surgery. We experienced a case of sudden death of a four-month-old male infant without congenital heart disease. It was elucidated by postmortem examination that the dead had suffered severe IE, which led him to death. In the microbiological genetic analysis using histological section, the pathogen causing inflammation in the present case was identified as Lactococcus lactis subspecies, although Staphylococci have been reported to be common and important one. Previously reported infectious diseases by Lactococcus lactis subspecies were all adult cases and this is the first report of an infantile death due to Lactococcal IE according to our knowledge. Any fatal disease may be included in sudden death cases targeted for forensic autopsy, even if it is rare. It is expected for forensic pathologists that they note such case and share each experience among themselves and other medical fields to develop a strategy for prevention.

Host-plant dependent population genetics of the invading weevil Hypera postica.

Population genetics of invading pests can be informative for understanding their ecology. In this study, we investigated population genetics of the invasive alfalfa weevil Hypera postica in Fukuoka Prefecture, Japan. We analyzed mitochondrial tRNALeu-COII, nuclear EF-1α gene fragments, and Wolbachia infection in relation to three leguminous host plants: Vicia angustifolia, Vicia villosa, and a new host Astragalus sinicus cultivated as a honey source and green manure crop. A parsimony network generated from mitochondrial gene sequences uncovered two major haplotypic groups, Western and Egyptian. In contrast to reported Wolbachia infection of the Western strain in the United States, none of our analyzed individuals were infected. The absence of Wolbachia may contribute to the stable coexistence of mitochondrial strains through inter-strain reproductive compatibility. Hypera postica genetic variants for the mitochondrial and nuclear genes were associated neither with host plant species nor with two geographic regions (Hisayama and Kama) within Fukuoka. Mitochondrial haplogroups were incongruent with nuclear genetic variants. Genetic diversity at the nuclear locus was the highest for the populations feeding on V. angustifolia. The nuclear data for A. sinicus-feeding populations indicated past sudden population growth and extended Bayesian skyline plot analysis based on the mitochondrial and nuclear data showed that the growth of A. sinicus-feeding population took place within the past 1000 years. These results suggest a shorter history of A. sinicus as a host plant compared with V. angustifolia and a recent rapid growth of H. postica population using the new host A. sinicus.

Structural characterization and chromosomal localization of the MAGE-E1 gene.

Genes of the melanoma-associated antigen (MAGE) family are characterized by the expression of tumor antigens on a malignant melanoma recognized by autologous cytolytic T lymphocytes. We have previously identified novel members of the MAGE gene family expressed in human glioma and named them MAGE-E1a-c. In the present study, we have revealed the genomic structure of MAGE-E1 by sequence analysis of a human chromosome bacterial artificial chromosome clone containing the MAGE-E1 gene. The MAGE-E1 gene is composed of 13 exons, and three of these (exon 2, exon 3 and exon 12) are alternatively spliced in each variant (E1a-c). The open reading frame encoding the MAGE-E1 peptides initiates in exon 2 and ends in exon 13. We have also demonstrated that the MAGE-E1 gene is located in Xp11 through the analysis of radiation hybrid panels. The genomic structure of MAGE-E1 is markedly similar to that of MAGE-D and its chromosomal locus is also identical to that of MAGE-D, but these features contrast with those of other MAGEs. These results suggest that MAGE-D and -E1 may be evolutionarily distant from other members of the MAGE family, and the two may be ancestral genes for the others.

Treatment of glioblastoma by direct inoculation of concentrated high titer-recombinant retrovirus carrying the herpes simplex virus thymidine kinase gene.

We prepared retroviruses carrying the lacZ gene or herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4-2.5 x 10(11) colony-forming units (cfu)/ml, and stereotaxically inoculated only 3 microliters of the retroviruses into a mouse glioma model. This resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty per cent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas. To achieve further safety in the gene therapy of glioma, genes abundantly expressed in human glioblastoma were searched by the Serial Analysis of Gene Expression (SAGE) technique. Among the top-147 most expressed tags in glioblastoma, we found a tag, TTTTGGGTAT, originated from an unidentified gene, which was not detected in human astrocyte cultures. Real-time quantitative RT-PCR showed that MAGE-E1 expression was 2.6-15 fold enriched in glioblastoma relative to human astrocytes. Expressed Sequence Tags (ESTs) containing this tag were homologous to melanoma-associated antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was cloned by 5'-rapid amplification of cDNA ends (RACE) technique. MAGE-E1 expression was enriched in glioblastoma and low in other cancers, and MAGE-E1 expression was detected only in brain and ovary among normal tissues. These results indicate that MAGE-E1 is a novel and glioma-specific member of MAGE family, which can be applied to glioma-specific gene transduction.

MAGE-E1, a new member of the melanoma-associated antigen gene family and its expression in human glioma.

To unearth glioma-specific genes in human glioblastoma, the serial analysis of gene expression technique was applied to a primary glioblastoma, using cultured human astrocytes as a normal control. Among the top 147 most-expressed tags in glioblastoma, we found a tag, TTTTGGGTAT, that originated from an unidentified gene and which was not detected in human astrocyte cultures. Real-time quantitative reverse transcription-PCR showed that MAGE-E1 expression was 2.6-15-fold enriched in glioblastoma relative to human astrocytes. Expressed sequence tags containing this tag were homologous to the melanoma-associated antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was cloned by the 5'-rapid amplification of cDNA ends technique. Three alternatively spliced variants (MAGE-E1a-c) were found, and deduced amino acid sequence showed that MAGE-E1a and -E1b shared the MAGE-conserved region, whereas -E1c did not. This suggests that although MAGE-E1c is expressed from one of the MAGE family, it has distinct functions from other members. Tissue distribution analysis showed that MAGE-E1 was distinct from other MAGEs. MAGE-E1 expression was detected only in brain and ovary among normal tissues. Interestingly, MAGE-E1a and/or -E1b were specifically expressed in glioma cells among cancer cells. These results indicate that MAGE-E1 is a novel and glioma-specific member of MAGE family.

A-type K+ current mediated by the Kv4 channel regulates the generation of action potential in developing cerebellar granule cells.

During neuronal differentiation and maturation, electrical excitability is essential for proper gene expression and the formation of synapses. The expression of ion channels is crucial for this process; in particular, voltage-gated K(+) channels function as the key determinants of membrane excitability. Previously, we reported that the A-type K(+) current (I(A)) and Kv4.2 K(+) channel subunit expression increased in cultured cerebellar granule cells with time. To examine the correlation between ion currents and the action potential, in the present study, we measured developmental changes of action potentials in cultured granule cells using the whole-cell patch-clamp method. In addition to an observed increment of I(A), we found that the Na(+) current also increased during development. The increase in both currents was accompanied by a change in the membrane excitability from the nonspiking type to the repetitive firing type. Next, to elucidate whether Kv4.2 is responsible for the I(A) and to assess the effect of Kv4 subunits on action potential waveform, we transfected a cDNA encoding a dominant-negative mutant Kv4.2 (Kv4.2dn) into cultured cells. Expression of Kv4.2dn resulted in the elimination of I(A) in the granule cells. This result demonstrates that members of the Kv4 subfamily are responsible for the I(A) in developing granule cells. Moreover, elimination of I(A) resulted in shortening of latency before the first spike generation. In contrast, expression of wild-type Kv4.2 resulted in a delay in latency. This indicates that appearance of I(A) is critically required for suppression of the excitability of granule cells during their maturation.

Completion of myelin compaction, but not the attachment of oligodendroglial processes triggers K(+) channel clustering.

The characteristic localization of ion channels is crucial for the propagation of saltatory conduction in myelinated nerves. Voltage-gated Na(+) channels are located at nodes of Ranvier while voltage-gated K(+) channels are mainly found at juxtaparanodal regions. Recently, a humoral factor secreted by oligodendrocytes has been reported to induce clustering of Na(+) channels in CNS axons. However, the molecular mechanisms for K(+) channel clustering as well as the role of oligodendrocytes are still uncertain. To clarify whether myelin sheath itself can induce the distinct distribution of K(+) channels, we have investigated the localization of K(+) channels in adult and developing mouse optic nerves. The CNS axons from chronic demyelinating and hypomyelinating mice were also examined to determine if myelin sheaths were required for the maintenance of clusters. In all cases, the K(+) channel clustering correlated well with compact myelin, but not with the presence of oligodendrocytes, suggesting that, in contrast to Na(+) channel clustering, the formation of compact myelin is required for initiation as well as maintenance of K(+) channel clustering. In addition, postsynaptic density protein-95 (PSD-95) or its highly related protein was found colocalized with K(+) channels, suggesting that it may interact with K(+) channels to form clusters at juxtaparanodal regions.

Expression of Kv3.1 and Kv4.2 genes in developing cerebellar granule cells.

The expression of voltage-gated potassium channels plays an important role in the acquisition of membrane excitability in neurons. We examined the expression pattern of genes in developing cerebellar granule neurons in vivo and in vitro. In situ hybridization of Kv3.1 mRNA demonstrated that the gene was expressed at high levels in the external granule layer (EGL) as well as in the internal granule layer (IGL) at all postnatal stages (P) examined (from P3 to P10). In contrast, Kv4.2 mRNA was detected in the premigratory zone (PMZ) of the EGL, but not in the proliferative zone (PLZ), in addition to the IGL. This indicates that Kv4.2 gene expression initiates in the postmitotic migrating neurons. We also examined the expression of the channel genes in microexplant culture systems. Kv3.1 polypeptide was detected in parallel fibers of granule cells at 2 days in vitro, and the expression continued in later stages. The signal of Kv4.2 protein was very low at 2 days in vitro; however, the number of positive cells and the intensity of the signals were increased at 6 days in vitro. These in vitro observations matched those in vivo and our previous electrophysiological studies in which we demonstrated that delayed- rectifier-type current was predominant in the immature granule cells followed by the later appearance of A-type current. The patterns of K(+) channel expression suggest that sequential expression of these channel genes primarily determines the membrane excitability.

Gene cloning and characterization of an acidic xylanase from Acidobacterium capsulatum.

The gene xynA encoding an acid endo-beta-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Eschrichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65 degrees C, and is stable pH between 3.0 and 8.0. The K(m) and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30 degrees C are 3.5 mg/ml and 403 mumol/min/mg.

Proximal promoter region is sufficient to regulate tissue-specific expression of UDP-galactose: ceramide galactosyltransferase gene.

UDP-galactose:ceramide galactosyltransferase (CGT) is the enzyme which catalyzes the final step of the biosynthesis of galactocerebroside (GalC), the most abundant glycolipid in myelin. We identified regulatory elements which are related to the tissue-specific expression of the mouse CGT gene by promoter assay using chimeric CGT-luciferase constructs. By comparing promoter activity in oligodendroglial CG4 cells and NIH3T3 fibroblasts, only a few hundred base pairs spanning from -309 to -98 were shown to be necessary for the tissue-specific activity of CGT promoter. A negative regulatory element was found in a more distal region, from -709 to -527, and it also worked in tissue-specific manner. Sequence analysis suggests that several known elements found commonly in myelin-related genes may explain these tissue-specific regulations of the transcriptional activity.

Differential interaction of voltage-gated K+ channel beta-subunits with cytoskeleton is mediated by unique amino terminal domains.

To define the molecular characteristics of K+ channel beta-subunit polypeptides, we have studied their biochemical properties and subcellular distribution in transfected mammalian cells. We find that the recombinant voltage-dependent K+ (Kv) beta1.1 and Kvbeta2 polypeptides have distinct detergent solubility properties owing to a novel association of Kvbeta1.1 with the actin-based cytoskeleton. Mutational and chimeric protein analyses show that the unique aminoterminus of Kvbeta1.1 is both necessary and sufficient for mediating the association of beta-subunits with cytoskeleton. Thus, the interaction with cytoskeleton is mediated through the amino-terminal domain previously shown to be necessary for modulating alpha-subunit inactivation, but not necessary for interaction with alpha-subunit polypeptides. These data reveal that different domains of beta-subunit polypeptides mediate interactions with cytoskeleton and with alpha-subunits, and provide a structural basis for previous reports that linked the extent of beta-subunit-induced inactivation to the state of the actin cytoskeleton.

Astrocytic lineage analysis by detection of GFAP promoter activity in vitro.

To survey the emergence and onset of differentiation of the astrocytic lineage in the developing mouse cerebral wall, the promoter activity of a 2.5 kb 5'-flanking region of glial fibrillary acidic protein (GFAP) was measured in individual developing brain cells using a retrovirus-mediated gene transfer system. We identified precursors for astrocytes in primary culture of embryonic mouse cerebral wall cells by detection of GFAP promoter activity, which was detected approximately 3 days prior to the appearance of GFAP immunoreactivity. Since retroviruses only integrate into the chromosomes of actively proliferating cells, cells detected by this method should have been mitotically active at the time of retroviral infection on day 15 postfertilization (E15). Furthermore, we observed that cells activating GFAP promoter were located near the ventricular surface of cultured cerebral wall slices as a cluster of spherical cells. These results demonstrate that precursor cells for astrocytes exist within the germinative zone of developing cerebral wall, and that these cells are mitotically active on day E15, which is a late stage of neuronal production period in the mouse cerebral wall. The morphology, location and mitotic activity of these cells suggest that they are unlikely to be cells that have been transformed from radial glial cells.

GFAP gene expression during development of astrocyte.

Glial fibrillary acidic protein (GFAP) is expressed exclusively in astrocytes in the central nervous system. In order to characterize individual cultured cells in which the GFAP promoter is active and to identify the regulatory mechanisms of GFAP expression in these cells, we have developed a unique assay system for promoter activity using retrovirus vectors. Retrovirus containing the mouse GFAP promoter fused to the lacZ gene were used to infect mixed glial cultures. The infected cells, in which the GFAP promoter was active, were visualized by X-Gal staining. From these experiments, we found that a 256 bp fragment 5' of the transcription initiation site was sufficient to confer astrocyte-specific expression of GFAP. The GFAP promoter became active about 3 days before GFAP protein can be detected immunohistochemically, which indicates that detection of GFAP promoter activity can be used to identify astrocyte progenitors. We have also established immortalized astrocyte cell lines in which we detect GFAP promoter activity. Immorto mouse is a transgenic mouse generated by the introduction of thermolabile SV40 T Ag, tsA58. A mixed glial culture prepared from 2-day-old Immorto mouse brain was incubated at 32 degrees C, at which temperature most of the cells expressed T Ag. The culture was then infected with retrovirus containing GFAP promoter-lacZ, and the infected cells were selected. Using the fluorescence-activated cell sorter with fluorescein di-beta-D-galactopyranoside as a substrate (FDG-FACS), these cells were separated into two groups: FDG(+), in which the GFAP promoter was active, and FDG(-), in which it was inactive. Mature astrocyte cell lines were established from the FDG(+) cells by colony isolation. The FDG(-) cells were cloned by colony isolation and cultured at 32 or 39 degrees C. At the latter temperature the expression of T Ag was suppressed and cell differentiation was induced in most cells. The cells which became positive for X-Gal staining only after switching to 39 degrees C were collected as immature astrocyte cell lines. These immortalized cell lines should be useful to investigate the molecular mechanisms of astrocyte differentiation.

Appearance of a fast inactivating voltage-dependent K+ currents in developing cerebellar granule cells in vitro.

To elucidate the molecular mechanisms that regulate the maturation of action potential, we began by examining voltage-dependent K+ currents, known to contribute to the maturation of action potential, of developing granule cells in mouse cerebellar microexplant cultures. The migration of developing granule cells in this culture is reported to mimic the in vivo process, but their specific identification is still incomplete. In this study, we identified and characterized granule cells in this culture. Immunocytochemical analysis found that granule cells migrated radially out from explants and subsequently formed small clusters and also that their morphology changed from a bipolar to a T shape during migration. Moreover, in the electrophysiological study, the GABA response of granule cells in this culture clarified that the electrophysiological properties of granule cells were normally maintained. We therefore have concluded, that this culture system is a powerful tool for investigating the differentiation of cerebellar granule cells. Based on these findings, we recorded voltage-dependent K+ currents of developing granule cells in this culture, while concurrently observing their morphology. Our results show that voltage-dependent K+ currents of developing granule cells change from delayed rectifier to A current in parallel with their morphological changes from bipolar to T-shaped cells.

Voltage-gated K+ channel beta subunits: expression and distribution of Kv beta 1 and Kv beta 2 in adult rat brain.

Recent cloning of K+ channel beta subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming alpha subunits. Here, we examined the expression, distribution, and association of two of these beta subunits, Kv beta 1 and Kv beta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta-subunit genes are heterogeneously expressed, with high densities of Kv beta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv beta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv beta 1 and Kv beta 2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kv beta 2 is the major beta subunit present in rat brain membranes, and that most K+ channel complexes containing Kv beta 1 also contain Kv beta 2. Taken together, these data suggest that Kv beta 2 is a component of almost all K+ channel complexes containing Kv 1 alpha subunits, and that individual channels may contain two or more biochemically and functionally distinct beta-subunit polypeptides.

Beta subunits promote K+ channel surface expression through effects early in biosynthesis.

Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.

Selective interaction of voltage-gated K+ channel beta-subunits with alpha-subunits.

To begin to study the molecular bases that determine the selective interaction of the beta-subunits of voltage-gated K+ channels with alpha-subunits observed in situ, we have expressed these polypeptides in transfected mammalian cells. Analysis of the specificity of alpha/bet a-subunit interaction indicates that both the Kvbeta1 and Kvbeta2 beta-subunits display robust and selective interaction with the five members of the Shaker-related (Kv1) alpha-subunit subfamily tested. The interaction of these beta-subunits with Kv1 alpha-subunits does not require the beta-subunit N-terminal domains. Thus, the previously observed failure of N-terminal mutants of Kv beta1 to modulate inactivation kinetics of Kv1 family members is not simply due to a lack of subunit interaction. Interaction of these beta-subunits with members of two other subfamilies (Shab- and Shaw-related) could not be detected. Somewhat surprisingly, a member of the Shal-related subfamily was found to interact with beta-subunits; however, this interaction had biochemical characteristics distinct from the beta-subunit interaction with Kv1 family members. In all cases, Kvbeta1 and Kvbeta2 exhibited indistinguishable alpha-subunit selectivity. These studies point to a selective interaction between K+ channel alpha- and beta-subunits mediated through conserved domains in the respective subunits.

Neuron-specific expression of a chicken gicerin cDNA in transient transgenic zebrafish.

Gicerin, a novel cell adhesion molecule which belongs to the immunoglobulin superfamily, is expressed temporally and spatially in the developing chick brain and retina. The previous in vitro experiments using transfected cells showed that gicerin can function as a cell adhesion molecule which has both homophilic and heterophilic binding activities. For the in vivo analyses of gicerin in neural development, we tried to utilize a zebrafish system, a vertebrate suitable for studying early development. We generated transient transgenic animals by microinjecting DNA constructs into zebrafish embryos. Chicken gicerin, under control of the neurofilament gene promoter, was preferentially expressed in neuronal cells and gicerin-expressing neurons exhibited a fasciculation formation with neighboring gicerin-positive axons, which may be partly due to homophilic cell adhesion activity of gicerin. These experimental results suggest that this fast and efficient transgenic animal system is useful for studying the functional roles of neuron-specific genes during the development.

A simplified general method for determination of recombinant retrovirus titers.

To construct recombinant retroviruses with only a single active promoter, we introduced point mutations into the TATA box region of the 3'-LTR, and successfully obtained high-titer virus with sufficient self-inactivating activity. However, the viral titer could not be determined by the number of G418 resistant colonies since the neomycin resistance gene was under 5'-LTR control, because of inactivation of the selection marker in target glioma cells. To overcome this problem, we constructed PCR primers with homology to a gene under the control of the internal promoter of recombinant retrovirus, and to retrovirus-specific sequences. There was good correlation between the amount of PCR-amplified product and the number of colony forming units when glioma cells were transduced with the retroviruses containing both the neomycin resistance gene and the HTK gene. Amplified PCR products quantitated by densitometry after glioma cells were transduced with SIV retrovirus vectors, and there was good correlation between density and sensitivity to GCV following transduction. Therefore, detection of HTK PCR products from glioma cells transduced with HTK-bearing retroviruses is useful for determining the appropriate packaging cell for efficient production of viral particles. This detection system is especially useful for isolating high titer clones producing SIV-type retroviruses.