A Single Amino Acid Substitution in the Intervening Region of 129K Protein of Cucumber Green Mottle Mosaic Virus Resulted in Attenuated Symptoms.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

Vascular architecture in the bacteriogenic light organ of Euprymna tasmanica (Cephalopoda: Sepiolidae).

Symbiosis between southern dumpling squid, Euprymna tasmanica (Cephalopoda: Sepiolidae), and its luminescent symbiont, the bacterium Vibrio fischeri, provides an experimentally tractable system to examine interactions between the eukaryotic host and its bacterial partner. Luminescence emitted by the symbiotic bacteria provides light for the squid in a behavior termed "counter-illumination," which allows the squid to mask its shadow amidst downwelling moonlight. Although this association is beneficial, light generated from the bacteria requires large quantities of oxygen to maintain this energy-consuming reaction. Therefore, we examined the vascular network within the light organ of juveniles of E. tasmanica with and without V. fischeri. Vessel type, diameter, and location of vessels were measured. Although differences between symbiotic and aposymbiotic squid demonstrated that the presence of V. fischeri does not significantly influence the extent of vascular branching at early stages of symbiotic development, these finding do provide an atlas of blood vessel distribution in the organ. Thus, these results provide a framework to understand how beneficial bacteria influence the development of a eukaryotic closed vascular network and provide insight to the evolutionary developmental dynamics that form during mutualistic interactions.

Dental findings in a child with osteopathia striata with cranial sclerosis (OS-CS): a case report.

The dental management of an 8-year-old girl with osteopathia striata with cranial sclerosis (OS-CS) is described. The girl presented with various oral abnormalities. The aim of this case report was to describe in detail the dental findings in a patient with OC-CS and the precautions to be taken when planning treatment. In the present case, many dental anomalies, such as delayed eruption of the permanent teeth, obliteration of the dental pulp, short roots, fused roots and taurodontism, were detected. In patients with OS-CS, routine dental care from an early stage is recommended to manage this anomaly properly.

Involvement of glucocorticoid receptor on hyperpyrexia induced by methamphetamine administration.

UNLABELLED: We have investigated the involvement of glucocorticoid on methamphetamine (MA) induced hyperpyrexia using a bio-telemetric system. A significant level of hyperpyrexia was observed in MA administered rats. In contrast, increase of body temperature was suppressed by adrenalectomy or by the administration of RU-486, an antagonist of the glucocorticoid receptor. These data suggest that the glucocorticoid receptor may be involved in hyperpyrexia induced by MA. KEYWORDS: methamphetamine - hyperpyrexia - glucocorticoid - corticosterone.

Phylogeographical patterns among Mediterranean sepiolid squids and their Vibrio symbionts: environment drives specificity among sympatric species.

Bobtail squid from the genera Sepiola and Rondeletiola (Cephalopoda: Sepiolidae) form mutualistic associations with luminous Gram-negative bacteria (Gammaproteobacteria: Vibrionaceae) from the genera Vibrio and Photobacterium. Symbiotic bacteria proliferate inside a bilobed light organ until they are actively expelled by the host into the surrounding environment on a diel basis. This event results in a dynamic symbiont population with the potential to establish the symbiosis with newly hatched sterile (axenic) juvenile sepiolids. In this study, we examined the genetic diversity found in populations of sympatric sepiolid squid species and their symbionts by the use of nested clade analysis with multiple gene analyses. Variation found in the distribution of different species of symbiotic bacteria suggests a strong influence of abiotic factors in the local environment, affecting bacterial distribution among sympatric populations of hosts. These abiotic factors include temperature differences incurred by a shallow thermocline, as well as a lack of strong coastal water movement accompanied by seasonal temperature changes in overlapping niches. Host populations are stable and do not appear to have a significant role in the formation of symbiont populations relative to their distribution across the Mediterranean Sea. Additionally, all squid species examined (Sepiola affinis, S. robusta, S. ligulata, S. intermedia, and Rondeletiola minor) are genetically distinct from one another regardless of location and demonstrate very little intraspecific variation within species. These findings suggest that physical boundaries and distance in relation to population size, and not host specificity, are important factors in limiting or defining gene flow within sympatric marine squids and their associated bacterial symbionts in the Mediterranean Sea.

A fatal case of severe methemoglobinemia presumably due to chlorate ingestion.

A fatal case due to severe methemoglobinemia is presented. A male in his forties was found unconscious in his house and, despite intensive care, death was confirmed approximately 11 hours later. Toxicological analysis using ion chromatography revealed the presence of chlorate in the stomach contents. However, chlorate was not detected in the blood, and no other drugs or ethanol were detected in the blood either. We concluded that the cause of death was presumably due to chlorate poisoning, based on the results of the autopsy and the toxicological examination.

BIOLOGICAL PROPERTIES (IN VITRO) EXHIBITED BY FREE-LIVING AND SYMBIOTIC VIBRIO ISOLATES.

Adhesion and biofilm forming ability of symbiotic bacteria play a crucial role in host colonization and tissue infection. Bacteria benefit by adhering to their host in a manner that allows them to successfully maintain contact for the exchange of nutrients, hormones, or other necessary products. This study examined pili morphology, motility, and biofilm formation exhibited by Vibrio fischeri strains (free-living and symbiotic). Since these symbiotic factors contribute in some fashion to the interaction between V. fischeri and their squid host, variation between strains may be a contributing factor that leads to specificity among different hosts. V. fischeri strains examined in this study demonstrated considerable variation in their biological properties when observed in vitro. In addition to differences observed between strains isolated from several different host species, we observed variation between strains isolated from the same host species from diverse geographical locations. This study suggests that subtle differences in the biological properties of closely related V. fischeri strains may influence the nature of the interaction among V. fischeri and their sepiolid hosts.

ULTRASTRUCTURE OF LIGHT ORGANS OF LOLIGINID SQUIDS AND THEIR BACTERIAL SYMBIONTS: A NOVEL MODEL SYSTEM FOR THE STUDY OF MARINE SYMBIOSES.

The class Cephalopoda (Phylum Mollusca), encompassing squids and octopuses, contains multiple species that are characterized by the presence of specialized organs known to emit light. These complex organs have a variety of morphological characteristics ranging from groups of simple, light-producing cells, to highly specialized organs (light organs) with cells surrounded by reflectors, lenses, light guides, color filters, and muscles. Bacteriogenic light organs have been well characterized in sepiolid squids, but a number of species in the family Loliginidae are also known to contain bacteriogenic light organs. Interest in loliginid light organ structure has recently arisen because of their potential as ecological niches for Vibrio harveyi, a pathogenic marine bacterium. This also implies the importance of loliginid light organs as reservoirs for V. harveyi persistence in the ocean. The present study utilized transmission and scanning electron microscopy to characterize the morphology of loliginid light organs and determined the location of bacterial symbiont cells within the tissue. It was determined that the rod-shaped loliginid symbionts lack flagella, as similarly observed in other light organ-associated bacteria. Also, the interaction of individual cells to light organ tissue is not as defined as reported for other squid-Vibrio systems. In addition, SEM observations show the presence of two pores leading to the bacterial chamber. Data presented here offer support for the hypothesis of environmental transfer of bacterial symbionts in loliginid squids.

THE EVOLUTIONARY ECOLOGY OF A SEPIOLID SQUID-VIBRIO ASSOCIATION: FROM CELL TO ENVIRONMENT.

Mutualistic relationships between bacteria and their eukaryotic hosts have existed for millions of years, and such associations can be used to understand the evolution of these beneficial partnerships. The symbiosis between sepiolid squids (Cephalopoda: Sepiolidae), and their Vibrio bacteria (gamma Proteobacteria: Vibrionaceae), has been a model system for over 20 years, giving insight as to the specificity of the association, and whether the interactions themselves give rise to such finely tuned dialog. Since the association is environmentally transmitted, selection for specificity can evolve from a number of factors; abiotic (temperature, salinity), as well as biotic (host species, receptors, cell/cell interactions). Here, we examine the transition between these forces effecting the symbiosis, and pose possible explanations as to why this association offers many attributes for understanding the role of symbiotic competence.

Deciphering Evolutionary Mechanisms Between Mutualistic and Pathogenic Symbioses.

The continuum between mutualistic and pathogenic symbioses has been an underlying theme for understanding the evolution of infection and disease in a number of eukaryotic-microbe associations. The ability to monitor and then predict the spread of infectious diseases may depend upon our knowledge and capabilities of anticipating the behavior of virulent pathogens by studying related, benign symbioses. For instance, the ability of a symbiotic species to infect, colonize, and proliferate efficiently in a susceptible host will depend on a number of factors that influence both partners during the infection. Levels of virulence are not only affected by the genetic and phenotypic composite of the symbiont, but also the life history, mode(s) of transmission, and environmental factors that influence colonization, such as antibiotic treatment. Population dynamics of both host and symbiont, including densities, migration, as well as competition between symbionts will also affect infection rates of the pathogen as well as change the evolutionary dynamics between host and symbiont. It is therefore important to be able to compare the evolution of virulence between a wide range of mutualistic and pathogenic systems in order to determine when and where new infections might occur, and what conditions will render the pathogen ineffective. This perspective focuses on several symbiotic models that compare mutualistic associations to pathogenic forms and the questions posed regarding their evolution and radiation. A common theme among these systems is the prevailing concept of how heritable mutations can eventually lead to novel phenotypes and eventually new species.

Bilateral brachial pull-through technique for stenting in a patient with stenosis of the vertebral artery origin: technical case report.

Stenting for stenosis of the proximal vertebral artery (VA) is commonly performed via a femoral approach. However, iliofemoral occlusive disease such as arteriosclerosis obliterans sometimes prevents safe transfemoral access. In certain situations where both femoral access and ipsilateral brachial access are difficult because of a concomitant vascular diseases or particular anatomic setting, a contralateral brachial approach using the brachiobrachial pull-through technique may allow efficient and accurate stenting. A case of VA origin symptomatic stenosis successfully treated with stenting using the new pull-through technique from the contralateral brachial artery to the brachial artery on the affected side is described.

Attenuated strains of tobacco mosaic virus. Reduced synthesis of a viral protein with a cell-to-cell movement function.

Attenuated strains of tobacco mosaic virus (TMV) have been used to protect crops against virulent strains. The synthesis of viral proteins and RNAs was investigated in protoplasts that had been infected separately with three tomato strains of TMV, virulent type L, and attenuated strains L11 and L11A. It was revealed that the mutations, which are responsible for the viral attenuation and have been mapped in the p126 (p184) gene, caused a reduction of the synthesis of the viral-coded p30 protein with a cell-to-cell movement function and its mRNA, but it had no significant effect on the synthesis of other viral proteins and RNAs in virus-infected protoplasts. Thus, it was shown that the attenuated strains can multiply as efficiently as the virulent strain in initially inoculated cells, but they can not spread efficiently outside the infected cells. In addition, it is suggested that a non-structural protein, p126 or p184, of TMV is involved in the synthesis of viral subgenomic p30 mRNA.

Quantitative immunoelectron-microscopic analysis of the type IV collagen alpha1-6 chains in the glomerular basement membrane in childhood thin basement membrane disease.

AIM: Thin basement membrane disease (TBMD) is characterized histologically by diffuse thinning of glomerular basement membrane (GBM). Although recent genetic analysis has shown that TBMD might be included within type IV collagen disorders, conventional immunohistochemical studies demonstrated normal labeling of type IV collagen alpha chains in the GBM. We have, however, successfully used confocal laser scanning microscopy to demonstrate a significantly reduced signal of type IV collagen alpha5 chain (alpha5(IV)) along capillary walls in TBMD. In order to further understand the association of type IV collagen with TBMD, we used immunoelectron microscopy to examine renal biopsies from 6 children with TBMD and six control children with minimal change nephrotic syndrome. METHODS: Ultrathin sections of LR gold resin were incubated with a rat monoclonal antibody against human alpha1(IV), alpha2(IV), alpha3(IV), alpha4(IV) alpha5(IV) or alpha6(IV) followed by colloidal gold conjugated goat anti-rat IgG. After taking electron micrographs, the labeling was quantitatively evaluated in the area occupied by the segments of basement membrane. The basement membrane was divided into three equal segments viz. subepithelial side, central portion and subendothelial side. RESULTS: In control subjects, the number of gold particles for alpha1(IV) or alpha2(IV) was significantly greater in the subendothelial side and central portion than in the subepithelial side of the GBM, whilst alpha3(IV), alpha4(IV) or alpha5(IV) labeling was significantly more prominent in the central portion compared to the subepithelial and subendothelial side of the GBM. TBMD samples showed a similar distribution pattern except that the subepithelial side and central portion of the GBM had a significantly reduced amount of alpha5(IV) antigen compared to control subjects. CONCLUSION: This is the first report demonstrating a diminished labeling intensity of alpha5(IV) in the central portion and subepithelial side of the GBM in renal biopsy specimens from patients with TBMD. These findings suggest that an abnormality of alpha5(IV) might possibly be associated with the pathogenesis of TBMD.

Mitochondrial sequence migrated downstream to a nuclear V-ATPase B gene is transcribed but non-functional.

A promiscuous nuclear sequence containing a mitochondrial DNA fragment was isolated from rice. Nucleotide sequence analysis reveals that the cDNA clone #21 carries a mitochondrial sequence homologous to the 3' portion of the rps19 gene followed by the 5' portion of the rps3 gene. The mitochondrial sequence is present in an antisense orientation. Sequence comparison of the #21 cDNA with the original mitochondrial sequence shows 99% similarity, suggesting a recent transfer event. Moreover, evidence for a lack of an RNA editing event and retaining of the group II intron sequence strongly suggests that the sequence was transferred from mitochondrion to the nucleus via DNA rather than RNA as an intermediate. The upstream region to the mitochondria-derived sequence shows homology to part of the vacuolar H(+)-ATPase B subunit (V-ATPase B) gene. Isolation of a functional V-ATPase B cDNA and its comparison with the #21 cDNA reveal a number of nucleotide substitutions resulting in many translational stop codons in the #21 cDNA. This indicates that the #21 cDNA sequence is not functional. Analysis of genomic sequences shows the presence of five intron sequences in the #21 cDNA, whereas the functional V-ATPase B gene has 14 introns. Of these, three exons and their internal two introns are homologous to each other, suggesting a duplication event of V-ATPase B genomic DNA. The results of this investigation strongly suggest that the mitochondrial sequence was integrated in an antisense orientation into the pre-existing V-ATPase B pseudogene that can be transcribed and spliced. This represents a case of unsuccessful gene transfer from mitochondrion to the nucleus.

Studies by site-directed mutagenesis of the carbohydrate-binding properties of a bark lectin from Robinia pseudoacacia.

A bark lectin, RBL, from Robinia pseudoacacia (black locust), binds galactose-related sugars specifically. Recombinant RBL (rRBL) with a histidine tag was expressed in Escherichia coli, purified and characterized. rRBL agglutinated rabbit erythrocytes and the hemagglutination was inhibited by galactose and related sugars. To elucidate the mechanism of the binding of carbohydrate by RBL, 16 mutant rRBLs were produced by site-directed mutagenesis. The analysis of the mutants indicated that residues Phe130 and Asp87 play key roles in the binding of carbohydrate by RBL. When Thu215, Leu217 and Ser218 in the carboxy-terminal region were replaced by alanine, the respective replacements decreased the hemagglutinating activity. However, replacement by alanine of Glu219 did not decrease this activity. Three mutant rRBLs were generated by reference to the primary sequences of the proposed carbohydrate- and metal-binding regions of mannose-specific lectins. Although these rRBLs agglutinated rabbit erythrocytes, the hemagglutination was not inhibited by mannose. Substitution or insertion that yielded a partial sequence similar to those of L-fucose-specific lectins and hemagglutinin from Maackia amurensis resulted in a complete loss of the hemagglutinating activity of rRBL.

Possible in vivo crosstalk between transcription factors with zinc-finger and leucine-zipper motifs in murine peripheral but not central excitable tissues.

In eukaryotes, de novo synthesis of proteins is mainly under control at the level of gene transcription by nuclear transcription factors with unique protein motifs such as leucine-zipper and zinc-finger. Binding of radiolabeled oligonucleotide probes for the "leucine-zipper" transcription factors, including activator protein-1 (AP1) and cyclic AMP response element binding protein (CREB), was markedly reduced in nuclear extracts of the adrenals from mice sacrificed 2 h after the subcutaneous injection of triamcinolone acetonide (TA), an agonist at glucocorticoid (GC) receptors which are also a transcription factor with "zinc-finger" motifs. The reduction was most significant 2 h after the administration, with recovery to the control level within 7 h after the injection. Moreover, the administration of TA invariably doubled immunoreactivities to an antibody against human GC receptors in nuclear fractions of the adrenal, pituitary and hypothalamus, with a concomitant reduction of those in cytosol fractions. Similar inhibition by TA was also seen with AP1 binding in the pituitary, while TA did not affect binding of radioprobes for AP1 and CREB in any discrete brain structures. These results suggest that systemic TA signals may be preferentially transduced into cell nuclei to attenuate DNA binding activities of AP1 through molecular mechanisms associated with crosstalk between transcription factors with different protein motifs in murine peripheral but not central excitable tissues.

The tomato mosaic tobamovirus movement protein interacts with a putative transcriptional coactivator KELP.

Viral movement through plasmodesmata in host plants likely depends on the interaction between virus-encoded movement protein (MP) and host proteins. In order to search for MP-interacting protein (MIP), we carried out far-western screening of a Brassica campestris cDNA library using a recombinant MP of tomato mosaic tobamovirus (ToMV) as a probe. One of the positive clones, designated MIP102, was found to be a putative orthologue for a transcriptional coactivator KELP of Arabidopsis thaliana. In vitro analysis with recombinant proteins revealed that ToMV MP could bind to KELP proteins that are derived from different plant species. At least 31 amino acids from the carboxyl-terminus of ToMV MP were dispensable for the interaction with KELP. Other MPs, derived from crucifer tobamovirus CTMV-W and cucumber mosaic cucumovirus, also exhibited comparable binding abilities. This suggests that these MPs could commonly interact with KELP, possibly to modulate the host gene expression.

A combined approach to the phylogeny of Cephalopoda (Mollusca).

Cephalopoda represents a highly diverse group of molluscs, ranging in habitat from coastal regions to deep benthic waters. While cephalopods remain at the forefront of modern biology, in providing insight into fields such as neurobiology and population genetics, little is known about the relationships within the group. This study provides a comprehensive phylogenetic analysis of Cephalopoda (Mollusca) using a combination of molecular and morphological data. Four loci (three nuclear 18S rRNA, fragments of 28S rRNA and histone H3 and one mitochondrial cytochrome c oxidase subunit I) were combined with 101 morphological characters to test the relationships of 60 species of cephalopods, with emphasis within Decabrachia (squids and cuttlefishes). Individual and combined data sets were analyzed using the direct optimization method, with parsimony as the optimality criterion. Analyses were repeated for 12 different parameter sets accounting for a range of indel/change and transversion/transition cost ratios. Most analyses support the monophyly of Cephalopoda, Nautiloidea, Coleoidea and Decabrachia, however, the monophyly of Octobrachia was refuted due to the lack of support for a Cirroctopoda + Octopoda group. When analyzing all molecular evidence in combination and for total evidence analyses, Vampyromorpha formed the sister group to Decabrachia under the majority of parameters, while morphological data and some individual data sets supported a sister relationship between Vampyromorpha and Octobrachia. Within Decabrachia, a relationship between the sepioids Idiosepiida, Sepiida, Sepiolida and the teuthid Loliginidae was supported. Spirulida fell within the teuthid group in most analyses, further rendering Teuthida paraphyletic. Relationships within Decabrachia and specifically Oegopsida were found to be highly parameter-dependent.

Analysis of macrophages in urine sediments in children with IgA nephropathy.

AIM: Although infiltrating macrophages found in renal biopsy specimens have been accepted as a useful marker for evaluating the activity of IgA nephropathy (IgAN), it is difficult to perform renal biopsies repeatedly, especially in children. To establish a more convenient and noninvasive method for estimating the degree of macrophage infiltration we examined the number of macrophages in urinary sediments. PATIENTS AND METHODS: Ten ml of morning urine were collected from 30 children with IgAN, 10 with thin basement membrane disease (TBMD), 8 with idiopathic renal hemorrhage (IRH) which was defined as nonglomerular hematuria due to nutcracker phenomenon revealed on ultrasonography, and 10 healthy children as controls. Ten of the 30 children with IgAN were treated with combination therapy comprising prednisolone, warfarin and dipyridamole and urine samples were collected weekly during the period of treatment. Two microl of the urine sediment were smeared on glass slides, dried and stained with a monoclonal antibody to human macrophages (anti-CD68, PG-M1) followed by a FITC-conjugated secondary antibody. After staining with propidium iodide (PI), the cells were examined by fluorescence microscopy with cells stained with both FITC and PI being counted as macrophages. In addition, anti-CD68 staining was used to quantify macrophage infiltration in renal biopsies from the same group of IgAN patients. RESULTS: The number of urine macrophages in children with IgAN was significantly higher than in children with TBMD and IRH as well as the control group (p < 0.01), whereas that was similar among TBMD, IRH and healthy children. In IgAN, there was a significant correlation between urine macrophage number and the activity index (p < 0.01), proteinuria (p < 0.01) and urine WBC count (p < 0.01). In addition, there was also a significant correlation between urine macrophage number and glomerular (p < 0.05) as well as interstitial macrophage infiltration (p < 0.01). In children with IgAN who received combination therapy, urine macrophage number decreased significantly (p < 0.01) in the 1st week of treatment whilst the degree of proteinuria decreased significantly (p < 0.01) in the 4th week. CONCLUSION: Urinary macrophage number may represent a noninvasive and straightforward estimate of the pathological activity evident in renal biopsy specimens, and may also be a more sensitive indicator than proteinuria of the therapeutic effect of interventional treatments in childhood IgAN.

Homology-dependent virus resistance in transgenic plants with the coat protein gene of sweet potato feathery mottle potyvirus: target specificity and transgene methylation.

ABSTRACT Nicotiana benthamiana plants were transformed with the coat protein (CP) coding sequence and the 3' nontranslated region (NTR) of the severe strain of sweet potato feathery mottle potyvirus (SPFMV-S). Regenerated lines were screened for virus resistance using recombinant potato virus X (PVX) engineered to contain the sequence homologous to the transgene. Out of 19 transgenic lines, 7 showed virus resistance after inoculation by the recombinant PVX. In most of the resistant lines, relatively low steady-state accumulation of the CP gene mRNA and little or no protein products were observed, suggesting that the resistance was manifested by a post-transcriptional gene-silencing mechanism. The resistant lines could be divided into two groups according to the target specificity of the silencing mechanism; one group recognizing the 3' part of the transgene mRNA and the other not only the 3' part, but also the 5' and the central part of the transgene mRNA. Particular regions of the transgene corresponding to the RNA target in the resistant lines were differentially methylated compared with the transgene sequence in a susceptible line.