Biological and phylogenetic characterization of Fusarium oxysporum complex, which causes yellows on Brassica spp., and proposal of F. oxysporum f. sp. rapae, a novel forma specialis pathogenic on B. rapa in Japan.

Although the causal agent of yellows of Brassica rapa (turnip, pak choi, and narinosa) in Japan was reported in 1996 to be Fusarium oxysporum f. sp. conglutinans, this classification has remained inconclusive because of a lack of detailed genetic and pathogenic studies. Therefore, we analyzed the taxonomic position of this organism using Japanese isolates of F. oxysporum complex obtained from diseased individuals of various B. rapa subspecies. Phylogenetic analyses using partial sequences of the rDNA intergenic spacer region and the mating-type gene (MAT1-1-1alpha-box) showed that B. rapa and cabbage isolates belong to different monophyletic clades that separated at early evolutionary stages. Additionally, correlations were observed between the molecular phylogeny and the vegetative compatibility groups. Isolates from turnip, komatsuna, and narinosa (B. rapa group) did not show pathogenicity against cabbage or broccoli (B. oleracea group), although they caused severe symptoms on their original host species. In contrast, cabbage isolates had significantly higher (P = 0.05) virulence on B. oleracea than on B. rapa crops. Our results indicate that F. oxysporum complex isolates from B. rapa and B. oleracea are not only phylogenetically distinct but also differ in host specificity. Therefore, we propose a novel forma specialis, F. oxysporum f. sp. rapae, which causes yellows on B. rapa, including turnip, komatsuna, pak choi, and narinosa.

Nuclear-matter modification of decay widths in the phi-->e+e- and phi-->K+K- channels.

The invariant mass spectra of phi-->K+K- are measured in 12 GeV p+A reactions in order to search for the in-medium modification of phi mesons. The observed K+K- spectra are well reproduced by the relativistic Breit-Wigner function with a combinatorial background shape in three betagamma regions between 1.0 and 3.5. The nuclear mass number dependence of the yields of the K+K- decay channel is compared to the simultaneously measured e+e- decay channel for carbon and copper targets. We parameterize the production yields as sigma(A)=sigma0Aalpha and obtain alphaphi-->K+K- -alphaphi-->e+e- to be 0.14+/-0.12. Limits are obtained for the partial decay widths of the phi mesons in nuclear matter.

Evidence for in-medium modification of the phi meson at normal nuclear density.

Invariant mass spectra of e(+) e(-) pairs have been measured in 12 GeV p + A reactions to detect possible in-medium modification of vector mesons. Copper and carbon targets are used to study the nuclear-size dependence of e(+) e(-) invariant mass distributions. A significant excess on the low-mass side of the phi meson peak is observed in the low betagamma(= beta/square root(1-beta(2))) region of phi mesons (betagamma < 1.25) with copper targets. However, in the high betagamma region (betagamma > 1.25), spectral shapes of phi mesons are well described by the Breit-Wigner shape when experimental effects are considered. Thus, in addition to our earlier publications on rho/omega modification, this study has experimentally verified vector meson mass modification at normal nuclear density.

Experimental signature of medium modifications for rho and omega mesons in the 12 GeV p+A reactions.

The invariant mass spectra of e+e- pairs produced in 12 GeV proton-induced nuclear reactions are measured at the KEK Proton Synchrotron. On the low-mass side of the meson peak, a significant enhancement over the known hadronic sources has been observed. The mass spectra, including the excess, are well reproduced by a model that takes into account the density dependence of the vector meson mass modification, as theoretically predicted.

Occurrence of White Top of Pea Caused by a New Strain of Pseudomonas syringae pv. pisi.

A new bacterial disease has been observed on pea in Shizuoka prefecture, Japan, since 1981. The disease occurs in early autumn when pea plants grow vigorously. The disease is characterized by chlorosis and whitening of apical shoots, including leaflets, stipules, and young pods. Usually, these white top (WT) symptoms are associated with extensive water-soaked lesions on stems and on leaflets at the basal part of the diseased plants. Thirty-four bacterial isolates from WT plants were characterized and identified together with 16 strains of Pseudomonas syringae pv. pisi from common bacterial blight of pea. The bacteria were gram-negative rods, having one to six polar flagella. The results of LOPAT tests were + - - - +, showing that they belong to P. syringe. In stab inoculation on stems, the WT isolates produced WT symptoms with water-soaked spots 14 days after inoculation. The 16 P. syringae pv. pisi strains never induced WT symptoms and, on the contrary, caused the typical bacterial blight. WT isolates were not pathogenic on any other plants tested. Phenotypic properties differentiated WT isolates and P. syringae pv. pisi strains into two groups; one consists of WT isolates and P. syringae pv. pisi group A, the other is P. syringae pv. pisi group B. Two distinct fingerprint profiles were identified by repetitive sequence based-polymerase chain reaction. WT isolates and P. syringae pv. pisi group A belonged to the same fingerprint type in rep-PCR, whereas a distinct fingerprint was shown by strains of the P. syringae pv. pisi group B. We concluded that the WT isolates should be included in P. syringae pv. pisi as a distinct strain in symptom expression.

The roles of IL-4, IL-5 and mast cells in the accumulation of eosinophils during allergic cutaneous late phase reaction in mice.

Late phase allergic response has been implicated in the pathogenesis of allergic diseases. In the current study, we investigated the role of IL-4, IL-5 and mast cells in the development of cutaneous late phase reaction (LPR) in mice. Antigenic challenge of ears of ovalbumin (OVA)-immunized BALB/c mice caused a biphasic ear swelling peaking at 1 hr (immediate phase reaction; IPR) and 24 hr (LPR). Ear swelling in LPR was significantly suppressed by the treatment with anti-IL-4 monoclonal antibody (mAb) before antigen challenge. Local eosinophil accumulation during LPR, however, was not inhibited by anti-IL-4 mAb. Moreover, anti-IL-5 mAb had no effect on the swelling response though it significantly suppressed the local accumulation of eosinophils. Interestingly, mast cell-deficient mice (WBB6F1-W/Wv) developed LPR without exhibiting IPR, while the magnitude of ear swelling and local eosinophilia was significantly lower than in normal congenic mice (+/+ mice). The present findings show that IL-4 and IL-5 differently regulate the development of LPR, and that IgE-mediated mast cell activation is required for full response.

Early treatment with corticosteroids ameliorates proteinuria, proliferative lesions, and mesangial phenotypic modulation in adult diffuse proliferative IgA nephropathy.

Diffuse proliferative immunoglobulin A (IgA) nephropathy has the potential risk for end-stage renal disease. However, treatment of IgA nephropathy has not been well established. To determine whether early treatment with corticosteroids ameliorates the proliferative lesions of diffuse proliferative IgA nephropathy, we conducted a prospective, randomized, controlled trial. Inclusion criteria were as follows: duration of abnormal urinalysis results less than 36 months, proteinuria less than 1.5 g/d of protein, serum creatinine level less than 1.5 mg/dL, and mesangial cell proliferation or matrix accumulation involving more than 50% of glomeruli. Twenty-one patients were randomly assigned to two groups: the corticosteroid group and the antiplatelet group. After 1 year of treatment, repeated renal biopsy was performed in 19 patients. We evaluated glomerular filtration rate, blood pressure, proteinuria, and histological parameters, including light microscopic findings and staining of alpha-smooth muscle actin (alphaSMA), as a marker of myofibroblast-like cells and fibronectin EDA (EDA-FN) as an indicator of renal fibrosis. After 1 year of treatment, proteinuria significantly decreased in the corticosteroid group. Histological findings, such as mesangial cell proliferation, mesangial matrix accumulation, and cellular crescents, showed significant improvement in the corticosteroid group but not in the antiplatelet group. Expression of alphaSMA in glomeruli significantly decreased in the corticosteroid group but not in the antiplatelet group. EDA-FN did not change in either group. We conclude that early treatment with corticosteroids for adult diffuse proliferative IgA nephropathy is effective in reducing renal injury.

Cardiovascular effect of normalizing the hematocrit level during erythropoietin therapy in predialysis patients with chronic renal failure.

The optimal target hematocrit (Ht) level in recombinant human erythropoietin (rHuEPO) therapy remains controversial and has hardly been investigated in predialysis patients. We prospectively studied the regression of left ventricular hypertrophy (LVH) on echocardiography in nine predialysis patients with chronic renal failure after a partial correction (target Ht, 30%) and normalization (target Ht, 40%) of the Ht with rHuEPO treatment. Twenty-four-hour ambulatory blood pressure monitoring was also performed. The administration of rHuEPO significantly increased Ht to the target values. The rate of renal failure progression did not change during rHuEPO treatment for 12 months (Cr, from 6.2 +/- 2.0 to 5.5 +/- 2.1 mg/dL). The left ventricular mass index (LVMI) tended to decrease after a partial correction of anemia (Ht, 32.1% +/- 1.8%) at 4 months, whereas it tended to significantly decrease after normalization of Ht (Ht, 39.1% +/- 2.4%) at 12 months (baseline, 140.6 +/- 12.1 g/m2; partial correction, 126.9 +/- 10.0 g/m2; normalization, 111.2 +/- 8.3 g/m2). All patients had received antihypertensive medication before rHuEPO administration, and additional drugs were also required in four cases during the study. As a result, a good overall blood pressure control was obtained without any adverse effects on the circadian blood pressure rhythm. In conclusion, from the perspective of LVH regression, the normalization of Ht was found to be more effective than that associated with a partial correction of anemia during rHuEPO therapy.

Alternate splicing in human Na+-MI cotransporter gene yields differentially regulated transport isoforms.

myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+-myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent approximately 1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3'-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3' exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.

IPD-1151T (suplatast tosilate) inhibits interleukin (IL)-13 release but not IL-4 release from basophils.

The effect of suplatast tosilate (IPD-1151T), which is known to suppress interleukin (IL)-4 release from T cells, on the release of IL-4 and IL-13 from human peripheral basophils was investigated. Basophils were obtained from 16 mite-sensitive atopic asthmatic patients. IPD-1151T clearly inhibited the antigen-induced release of IL-13 but not IL-4. These results suggest that IPD-1151T possesses different activity for the regulation of cytokine release in basophils and T cells.

Cyclosporin A mono-therapy in nephrotic syndrome with contra-indication of steroid therapy.

We describe three cases of nephrotic syndrome with a contra-indication for steroid therapy successfully treated with cyclosporin A (CsA). A 21-year-old man with focal segmental glomerulosclerosis (FSGS) complicated by necrosis of the femoral head, and a 34-year-old woman and a 48-year-old man with minimal change disease (MCD) complicated by psychogenic reaction and diabetes mellitus, respectively, were given CsA at initial dosages of 3.8-5.0 mg/kg/day and immediately remitted completely. However, two of these patients suffered relapses when CsA was tapered. They are currently maintained in complete or partial remission on CsA at dosages of 3.2-4.7 mg/kg/day. These findings suggest that CsA mono-therapy may be useful in nephrotic syndrome patients contra-indicated for steroid therapy.

Effect of early oral fluoroquinolones in hemorrhagic colitis due to Escherichia coli O157:H7.

During the Sakai outbreak of Escherichia coli O157:H7 infection, which was linked to contaminated cafeteria school lunches, there were several treatment modalities with regard to antimicrobial drugs. Patient outcomes among three hospitals with different modalities were compared retrospectively. Hemolytic uremic syndrome did not develop in any of the 15 patients treated with oral fluoroquinolone therapy; however, HUS did develop in three of 15 patients treated with intravenous (i.v.) fosfomycin and in two of 12 patients treated with i.v. cefotaxime and oral fosfomycin. The results indicate that oral fluoroquinolone therapy administered within 3 days of illness is effective in preventing the development of HUS; however, prospective randomized double-blind studies on early antimicrobial therapy of O157 hemorrhagic colitis are necessary. Several antibiotics, including fluoroquinolones, were reported to induce the production or release of Shiga-like toxins (STX) from E. coli O157:H7 in vitro. Although patients were examined for fecal STX, no STX were detected in the stools of patients treated with oral fluoroquinolones. In fact, treatment with fluoroquinolones for 5 days eradicated E. coli O157 in all patients.

Encephalopathy and cytomegalovirus colitis in an AIDS child.

A 2-year-old girl, who had prolonged thrush and spastic diplegia, was found to have a mother-to-child vertical transmission of human immunodeficiency virus type-1 (HIV). A brain computed tomography scan revealed a symmetrical calcification on the bilateral basal ganglia and periventricular white matter. She had an acquired immune deficiency syndrome (AIDS) encephalopathy of pure dominant pyramidal tract disorder without an intellectual deficit. Helper cell lymphocyte count (CD4) increased with the beginning of zidovudine (ZDV, also known as AZT) monotherapy but began to decrease after the 4th week to reach the baseline at 20th week. Zidovudine plus didanosine combination therapy was started at the 68th week, but because of intolerance, the combination was changed to ZDV plus lamivudine at the 98th week. By the 80th week, neither severe opportunistic infection nor deterioration of the neurological status was recognized, but chronic diarrhea appeared. The diarrhea advanced to the wasting syndrome at the age of 4 years and cytomegalovirus genome was confirmed in a biopsied specimen of the colon. Ganciclovir treatment was effective in stopping the diarrhea and increasing her bodyweight, but after the age of 5, resumption of diarrhea was followed by progressive emaciation and weakness. This work may provide some clues in treating children's AIDS.

Human Na(+)-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts.

Na(+)-myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myo-inositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na(+)-myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na(+)-dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and approximately 1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress.

Augmented expression of glomerular basement membrane specific type IV collagen isoforms (alpha3-alpha5) in experimental membranous nephropathy.

In human and experimental membranous nephropathy, new extracellular matrix accumulates between, and eventually surrounds, immune deposits on the subepithelial aspect of the glomerular basement membrane (GBM). To define the nature and source of this newly deposited matrix, we studied by in situ hybridization and immunohistology the production and tissue deposition of the recently defined basement membrane type IV collagen chain isoforms alpha3, alpha4, and alpha5, the mesangium-specific alpha1 and alpha2 isoforms of type IV collagen, and the fibrillar interstitial type I collagen during the development of immunological injury in passive Heymann nephritis (PHN), a rodent model of membranous nephropathy. Our results show that steady-state mRNA levels of alpha3-alpha5 (IV) but not alpha1 (IV) are significantly increased in the glomeruli of rats with PHN at the peak of immunological injury after 14 days. Increased signal for alpha4 (IV) and the new appearance of alpha1 (I) could be clearly localized to glomerular podocytes, the target of injury in this model. In addition, increased levels of immunoreactive alpha3-alpha5 were visible in the peripheral and paramesangial GBM together with de novo deposits of type I collagen. A modest increase in mesangial staining for alpha1/alpha2 (IV) was present in PHN glomeruli. In rats depleted of complement for 5 days after PHN induction, the peak of alpha4 (IV) mRNA expression on day 14 was blunted. In conclusion, we have shown increased production of the intrinsic GBM type IV collagen isoforms alpha3-alpha5 and ectopic production of type I collagen by injured podocytes in PHN. These changes may contribute to the formation of an expanded and disorganized GBM, as seen in experimental and human membranous nephropathy.

Mechanism of the progression of diabetic nephropathy to renal failure.

Although the evolution of diabetic nephropathy is brought about mostly by persistent hyperglycemia, its progression may be influenced by various other factors such as hypertension and dietary protein intake. It has been recently suggested in the literature that the gene polymorphism of angiotensin converting enzyme (ACE) might be associated with the development of diabetic nephropathy, because the DD genotype of ACE gene is closely associated with the presence of nephropathy in diabetic subjects. However, in our present analysis the frequency of the DD genotype in patients with non-insulin dependent diabetes is not significantly related to the presence or absence of nephropathy. It remains to be clarified by multi-center analysis using large numbers of patients whether the gene polymorphism of ACE is related to the progression of diabetic nephropathy to renal failure. Furthermore, it has been postulated that the interstitial fibrosis evaluated in renal biopsy specimens is significantly correlated with the declining of renal function in diabetic patients. However, it is not possible to clinically quantitate the interstitial fibrosis without performing renal biopsy. We have recently found that the urinary excretion of type IV collagen is significantly increased in diabetic patients. Moreover, the increase in urinary type IV collagen is well correlated with the amount of urinary albumin. Since type IV collagen in the urine is probably derived from tubulointerstitial tissue, it is likely that the increased amount of type IV collagen in the urine may reflect the fibrotic change in diabetic kidneys. Whether the increase in urinary type IV collagen is able to predict for the progression of diabetic nephropathy in the future should be examined.