Risk of haemolytic uraemic syndrome caused by shiga-toxin-producing Escherichia coli infection in adult women in Japan.

Shiga-toxin-producing Escherichia coli (STEC) infections usually cause haemolytic uraemic syndrome (HUS) equally in male and female children. This study investigated the localization of globotriaosylceramide (Gb3) in human brain and kidney tissues removed from forensic autopsy cases in Japan. A fatal case was used as a positive control in an outbreak of diarrhoeal disease caused by STEC O157:H7 in a kindergarten in Urawa in 1990. Positive immunodetection of Gb3 was significantly more frequent in female than in male distal and collecting renal tubules. To correlate this finding with a clinical outcome, a retrospective analysis of the predictors of renal failure in the 162 patients of two outbreaks in Japan was performed: one in Tochigi in 2002 and the other in Kagawa Prefecture in 2005. This study concludes renal failure, including HUS, was significantly associated with female sex, and the odds ratio was 4·06 compared to male patients in the two outbreaks. From 2006 to 2009 in Japan, the risk factor of HUS associated with STEC infection was analysed. The number of males and females and the proportion of females who developed HUS were calculated by age and year from 2006 to 2009. In 2006, 2007 and 2009 in adults aged >20 years, adult women were significantly more at risk of developing HUS in Japan.

Autosomal recessive cystinuria caused by genome-wide paternal uniparental isodisomy in a patient with Beckwith-Wiedemann syndrome.

Approximately 20% of Beckwith-Wiedemann syndrome (BWS) cases are caused by mosaic paternal uniparental disomy of chromosome 11 (pUPD11). Although pUPD11 is usually limited to the short arm of chromosome 11, a small minority of BWS cases show genome-wide mosaic pUPD (GWpUPD). These patients show variable clinical features depending on mosaic ratio, imprinting status of other chromosomes, and paternally inherited recessive mutations. To date, there have been no reports of a mosaic GWpUPD patient with an autosomal recessive disease caused by a paternally inherited recessive mutation. Here, we describe a patient concurrently showing the clinical features of BWS and autosomal recessive cystinuria. Genetic analyses revealed that the patient has mosaic GWpUPD and an inherited paternal homozygous mutation in SLC7A9. This is the first report indicating that a paternally inherited recessive mutation can cause an autosomal recessive disease in cases of GWpUPD mosaicism. Investigation into recessive mutations and the dysregulation of imprinting domains is critical in understanding precise clinical conditions of patients with mosaic GWpUPD.

A novel de novo point mutation of the OCT-binding site in the IGF2/H19-imprinting control region in a Beckwith-Wiedemann syndrome patient.

The IGF2/H19-imprinting control region (ICR1) functions as an insulator to methylation-sensitive binding of CTCF protein, and regulates imprinted expression of IGF2 and H19 in a parental origin-specific manner. ICR1 methylation defects cause abnormal expression of imprinted genes, leading to Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Not only ICR1 microdeletions involving the CTCF-binding site, but also point mutations and a small deletion of the OCT-binding site have been shown to trigger methylation defects in BWS. Here, mutational analysis of ICR1 in 11 BWS and 12 SRS patients with ICR1 methylation defects revealed a novel de novo point mutation of the OCT-binding site on the maternal allele in one BWS patient. In BWS, all reported mutations and the small deletion of the OCT-binding site, including our case, have occurred within repeat A2. These findings indicate that the OCT-binding site is important for maintaining an unmethylated status of maternal ICR1 in early embryogenesis.

Bilateral papillary renal cell carcinoma and angiomyolipoma in the patients with autosomal dominant polycystic kidney disease: case report of two cases and literature review.

We herein report two rare cases of bilateral renal neoplasms associated with autosomal dominant polycystic kidney disease (ADPKD). Case 1: Bilateral nephrectomy was performed on bilateral renal masses in a 58-year-old man with ADPKD. Case 2: Bilateral nephrectomy was performed on bilateral renal masses in a 32-year-old man with clinically suspected ADPKD. In case 1, angiomyolipoma (AML) and papillary renal cell carcinoma (PRCC) (type 1) were detected in the bilateral kidneys. In case 2, PRCC (type 1) was detected in the bilateral kidneys.

Bovine serum albumin-doxorubicin conjugate overcomes multidrug resistance in a rat hepatoma.

A bovine serum albumin-conjugated doxorubicin via the glutaraldehyde bridge (BSA-DXR conjugate) showed potent dose-dependent inhibition of cell growth against daunorubicin-resistant AH66 (AH66DR) cells as well as parental AH66 (AH66P) cells in vitro as compared to treatment with DXR or BSA-glutaraldehyde conjugate without DXR (BSA-GA). In the culture of AH66DR with BSA-DXR conjugate, drug accumulation in the AH66DR cells increased as a function of time up to 24 h reaching approximately the same drug level as AH66P cells treated with DXR. The intracellular accumulation of the BSA-DXR conjugate was inhibited by the addition of ammonium chloride, while that of DXR alone was not inhibited. Intracellular DXR was effluxed rapidly from AH66DR cells, but BSA-DXR conjugate or pharmacologically active DXR adduct remained in the cells at a relatively high concentration over a 36-h time period. The life-prolonging effect of the conjugate was assessed using rats inoculated i.p. with AH66P or AH66DR. The rats were treated with the BSA-DXR conjugate, DXR, a mixture of DXR with BSA, or BSA-GA by either the i.p. or i.v. route. Treatment with DXR had no significant surviving effect as compared to that with saline in AH66P-bearing rats. By contrast, BSA-DXR conjugate showed a significant life-prolonging effect as compared with DXR alone in the same degree both in AH66P- and AH66DR-bearing rats. BSA-GA did not show any toxicity in vivo as well as in vitro. These results indicate that the BSA-DXR conjugate allows DXR to escape from the multidrug resistance mechanism.

Studies on Escherichia coli chromosome proteins. II. DNA polymerases associated with the nucleoid.

Escherichia coli nucleoids isolated in the presence of spermidine have been shown to contain DNA polymerase activity of which more than 95% was ascribed to DNA polymerase I and the rest to DNA polymerases II and III. It also has been found that DNA polymerases II and III or DNA polymerase III substituted entirely for DNA polymerase I or DNA polymerase I and II in the nucleoids isolated from E. coli mutants lacking the corresponding enzyme activities.

Bidirectional Glenn procedure improves the mechanical efficiency of a total cavopulmonary connection in high-risk fontan candidates.

BACKGROUND: A total cavopulmonary connection (TCPC) is a widely performed surgical procedure for Fontan candidates. High-risk candidates who have undergone the bidirectional Glenn procedure (BDG) before TCPC have shown good results. The exact mechanism of this procedure, however, is still poorly understood. We hypothesized that a volume reduction with BDG improved ventricular contractility, thereby optimizing mechanical efficiency after TCPC. METHODS AND RESULTS: We measured percent normal systemic ventricular end-diastolic volume (%N-EDV), contractility (end-systolic elastance; E(es)), afterload (effective arterial elastance; E(a)), and mechanical efficiency (ventriculoarterial coupling; E(a)/E(es)) on the basis of the cardiac catheterization data before and after TCPC. Eighteen patients who underwent staged TCPC after BDG (staged group) were compared with 29 patients who underwent primary TCPC (primary group). E(es) and E(a) were approximated as follows: E(es)=mean arterial pressure/minimal ventricular volume, and E(a)=maximal ventricular pressure/(maximal ventricular volume-minimal ventricular volume), and E(a)/E(es) was then calculated. The ventricular volume was normalized with the body surface area. A canine experimental model with conductance catheter was used to validate the accuracy of this approximation of E(es) and E(a). %N-EDV decreased after TCPC in both groups. In the staged group, a smaller ventricular volume resulted in better contractility (E(es)). Although afterload (E(a)) increased in both groups, the increment of E(a) was smaller in the staged group. These changes resulted in an improvement of E(a)/E(es) in the staged group, whereas E(a)/E(es) increased in the primary group. CONCLUSIONS: The volume reduction of BDG preceding TCPC allows for any afterload mismatch to be corrected, thereby improving ventricular energetics after TCPC.

Expression of three mRNA species from a single rat aldolase A gene, differing in their 5' non-coding regions.

The complete nucleotide sequence of the rat aldolase A isozyme gene, including the 5' and 3' flanking sequences, was determined. The gene comprises ten exons, spans 4827 base-pairs and occurs in a single copy per haploid rat genome. The genomic DNA sequence was compared with those of three species of rat aldolase A mRNA (mRNAs I, II and III) that have been found to differ from each other only in the 5' non-coding region and to be expressed tissue-specifically. It revealed that the first exon (exon M1) encodes the 5' non-coding sequence of mRNA I, while the second exon (exon AH1) encodes those of mRNAs II and III and the following eight exons (exons 2 to 9) are shared commonly by all the mRNA species. These results allowed us to conclude that mRNA I and mRNAs II, III were generated from a single aldolase A gene by alternative usage of exon M1 or exon AH1 in addition to exons 2 to 9. S1 nuclease mapping of the 5' ends of their precursor RNAs suggested that these three mRNA species were transcribed from three different initiation sites on the single gene.

Sequence-based structural features between Kvlqt1 and Tapa1 on mouse chromosome 7F4/F5 corresponding to the Beckwith-Wiedemann syndrome region on human 11p15.5: long-stretches of unusually well conserved intronic sequences of kvlqt1 between mouse and human.

Mouse chromosome 7F4/F5 is a syntenic locus of human 11p15.5 in which many imprinted genes are clustered. Transmission of aberrant human 11p15.5 or duplicated 11p causes Beckwith-Wiedemann syndrome (BWS) depending on which parent the chromosome is derived from. To analyze a syntenic mouse locus corresponding to human 11p15.5, mouse BAC contigs were constructed between Nap2 and Tapa1, in which 390 kb was sequenced between Kvlqt1 and Tapa1. An unexpected finding was that of highly conserved intronic sequences of Kvlqt1 between mouse and human, and their homologies came up to at least 160 kb because the length of this gene extended to 350 kb, suggesting the possibility of some functional constraint due to transcriptional and/or post-transcriptional regulation of this region. Many expressed sequence tags (ESTs) were mapped on this locus. Three genes, Lit1 (Kvlqt1-AS), Mtr1 and Tssc4, were identified and characterized. Lit1 is an antisense-transcript of Kvlqt1 and paternally expressed and maternally methylated throughout the developmental stage. The position where Lit1 exists corresponded to a highly conserved region between mouse and human. This transcript extends at least 60 kb from downstream to upstream of exon 10 in Kvlqt1. Tssc4 and Mtr1 carried putative open reading frames but neither was imprinted. Further characterization of this locus based on the sequence comparison between mouse and human will contribute valuable information towards resolving the mechanism of the occurrence of BWS and the associated childhood tumor.

Rat aldolase A messenger RNA: the nucleotide sequence and multiple mRNA species with different 5'-terminal regions.

The nucleotide sequence of aldolase A mRNA in rat skeletal muscle was determined using recombinant cDNA clones and a cDNA synthesized by primer extension. The sequence is composed of 1343 nucleotides (nt) except for the poly(A) tail. Based on the sequence analysis we have deduced an open reading frame with 363 amino acids (aa) (Mr 39134). The sequence suggests several nt polymorphisms in the mRNA population, one of which causes an aa change. The determined sequence of rat aldolase A mRNA was compared with the published ones of rabbit aldolase A or rat aldolase B mRNAs. The homology between rat and rabbit aldolase A mRNA sequences is greater than that between rat aldolase A and B mRNA sequences. Multiple aldolase A mRNAs having different Mrs were detected in the various tissues, and appeared to be expressed in a tissue-specific manner. Further analysis suggests that differences in mRNA length are due to differences in the 5'-noncoding terminal region.

C11orf21, a novel gene within the Beckwith-Wiedemann syndrome region in human chromosome 11p15.5.

A novel gene, C11orf2, was identified by BLAST search in the human chromosome 11p15.5 region potentially responsible for Beckwith-Wiedemann Syndrome (BWS) and some cancers. Two cDNA clones with different sizes were obtained, which share a potential ORF of 399bp and are different in their 3' untranslated regions. This gene was revealed to be expressed exclusively in human heart and in almost no other tissues examined by northern blotting. Two transcripts of different sizes, 0.9 and 3.1kb, were identified in heart, consistent with the length of the two cDNA clones. The gene shows biallelic expression (non-imprinted) in fetal liver, although it is located in the imprinted domain of 11p15.5. C11orf21 codes a protein of 132 amino acids as proved by the expression of C11orf21-EGFP fusion protein in cultured cells. The EGFP-fusion protein expressed in cultured cells localized mainly in the cytoplasm.

Analysis of upstream regulatory regions required for the activities of two promoters of the rat aldolase A gene.

Rat aldolase A gene has 2 promoters with different tissue specificities (M- and AH promoters). The M promoter is active only in adult skeletal muscle and induced during myogenesis, whereas the AH promoter is active ubiquitously in many tissues, including various cancer cells. Regulatory sequences for these promoters were investigated through assays for transient expression after introduction into myogenic and nonmyogenic cells. When M promoter-CAT fusion genes were transfected into primary cultures of chicken myoblasts, expression of CAT activity was drastically induced during myotube formation. The region comprising 202 to 85 base pairs (bp) upstream from the transcription initiation site was found to be necessary for the induction and an enhancer activity whose region includes the AT-rich recognition sequence (MEF-2 binding site). On the other hand, 2 upstream regions were found to be responsible for AH promoter activity expressed in HepG2 cells. The distal region (-280 to -260) of the promoter includes the AP1 binding sequence, whereas the proximal region (-207 to -180) contains a novel inverted repeat consisting of 22 bp but does not contain known promoter and enhancer sequences.

A monoclonal antibody CF703 raised against human fetal lung recognizes a novel onco-fetal mucin.

To obtain murine monoclonal antibodies (MAb), mice were immunized with the extract of a human fetal lung from the first trimester of gestation as initial immunogen followed by booster immunization with a human lung cancer cell line. MAb CF703 which reacted to the booster material was screened and selected. In adults, expression of the antigen defined by MAb CF703, CF703 antigen, was shown immunohistochemically in 78% of gastric carcinomas (14/18), 59% of ovarian adenocarcinomas (10/17), 50% of pancreatic carcinomas (5/10), 17% of lung carcinomas (4/24), 50% of uterine cervical adenocarcinomas (2/4), 20% of endometrial adenocarcinomas (1/5) and 10% of renal cell carcinomas (1/10). Other malignant tumors failed to demonstrate the reactivity. MAb CF703 was weakly reactive with only three adult normal tissues including surface lining of gastric mucosa, Brunner's glandular epithelia of the duodenum and columnar epithelia in the uterine endocervix. In fetal tissues, 5 of 23 tissues including squamous epithelia in lower portion of the;esophagus, surface epithelia of gastric mucosae and goblet cells in the small and large intestine were reactive. The CF703 antigen had a molecular weight over 500 kDa and its epitope was carbohydrate in nature by reason of the resistance to proteinase digestion and sensitivity to periodate oxidation, neuraminidase and alkaline-borohydrite. MAb CF703 recognizes probably a unique epitope in oncodevelopmental mucin glycoprotein. Additionally, this immunization method has the advantage of rapid acquirement of MAbs with restricted, narrow cancer spectrum and is a worthy strategy for generating the new MAbs.

Monoclonal-antibodies 5g8 and 2h6 are complementary immunohistochemical markers of lung carcinomas.

Two murine monoclonal antibodies (MAbs) 5G8 and 2H6 were generated by fusing spleen cells obtained from mice immunized with human fetal tissue extract (14 weeks, Nonidet P-40 membrane fraction) from the early first trimester, followed by a booster injection of a lung cancer cell line. The MAb 5G8 recognized antigens with a molecular weight of 90, 50, 20 kDa, was neuraminidase-resistant and showed cross-reactivity with both carcinoembryonic antigen and non-specific cross-reacting antigen. The MAb 2H6 reacted with a sialomucin whose molecular weight was more than 1000 kDa and was different from previously known tumor associated-marker antigens. The distribution of the MAb-recognizing antigens in various tissues was investigated immunohistochemically. In malignant epithelial tumors of the lung, acinar adenocarcinoma and bronchioloalveolar carcinoma were positive for both MAbs; papillary adenocarcinoma, squamous cell carcinoma, and adenoid cystic carcinoma were positive only for MAb 5G8; solid carcinoma with mucin formation, small cell carcinoma, and large cell carcinoma were positive only for MAb 2H6. The combination of the two MAbs covered almost all histological types of lung carcinomas (23 of 24 cases) except carcinoid tumors. MAbs 5G8 and 2H6 reacted also with a restricted number of adenocarcinomas of the other organs. MAb 5G8 did not react with normal fetal or adult tissues, except for metaplastic gastric mucosa, acinar cells of the breast and leucocytes, whereas MAb 2H6 reacted with the surface epithelium of the stomach and colon, the Brunner gland of the duodenum and uterine cervix as well as the epithelium of the fetal digestive tract. Thus, MAb 2H6 which recognized oncofetal sialomucin, played a complementary role to MAb 5G8 as a CEA-related MAb in the immunohistochemical diagnosis of lung carcinomas.

Doxorubicin enhances transient expression of p-glycoprotein and modulates activity and isoform expression of protein-kinase-C in ah66 rat hepatoma-cells.

Intracellular changes in enzyme activity and the isoform pattern of protein kinase C (PKC) during treatment with doxorubicin (DXR) were examined in a rat ascites hepatoma AH66 parental cell line (AH66P), which shows slight expression of P-glycoprotein (Pgp) on the cell membrane and is classified as an intrinsic multidrug-resistant cell. AH66P cells, treated for 10 days with DXR at the concentrations of 0.3 or 5.0 muM, exhibited moderate to severe inhibition of PKC activity after transient increase of the activity, which mainly was either Ca2+-independent and phospholipid-dependent or Ca2+-independent and phospholipid-independent. PKC isoform analysis of the treated cells also indicated the transient 1.5- and 1.8-fold increase of PKC-delta and PKC-zeta as well as slight increase of PKC-alpha in treatment with 0.3 muM DXR and 1.9- and 1.6-fold increase of PKC-alpha and PKC-zeta with slight increase of PKC-delta in treatment with 5.0 muM, respectively. A small, but interesting discrepancy between the markedly elevated enzyme activity, which was composed particularly of both cofactor-independent forms, and the relatively low levels of isoform expression, was found when the cells were treated with 5.0 muM DXR. It is of great interest that DXR-treatment, while only slight increasing P-glycoprotein (Pgp) phosphorylation, enhanced the transient expression of Pgp on the cell membrane with good correlation with the fluctuating change in PKC activity. These results indicate that the possible expression of some or novel specific isoform(s) of PKC may modulate and be associated with expression of the Pgp.

Monoclonal antibody, Mab 12C3, is a sensitive immunohistochemical marker of early malignant change in epithelial ovarian tumors.

A murine monoclonal antibody (MAb 12C3) that is specific to human ovarian carcinomas was generated by immunizing mice with a human ovarian germinoma cell line (JOHYC-2). The antigen distribution that was defined by MAb 12C3 in normal and malignant human tissues was analyzed by immunohistochemistry on paraffin-embedded and frozen sections. The antibody reacted with 67.7% (21 of 31 cases) of epithelial ovarian carcinomas (6 of 12 cases of serous cystadenocarcinoma, 5 of 7 cases of mucinous cystadenocarcinoma, 7 of 9 cases of clear cell carcinoma, 3 of 3 cases of endometrioid adenocarcinoma), but did not react with any of the benign epithelial ovarian adenomas tested. Partial regions of borderline ovarian malignancies that exhibited marked papillary projection of the lining cells with cellular atypia reacted positively with MAb 12C3 in 14 of 25 cases (56.0%). The histologic features of regional early malignant change corresponded to the expression of the MAb 12C3 epitope in the borderline malignant tumor cells. There was a low frequency of reaction (4.3%) between the antibody and other gynecologic and nongynecologic malignancies (46 cases of 12 tissues). In normal tissues, the antibody reacted positively with only three tissues, including corpora lutein cells, excretory ducts in the submandibular gland, and basal cells of the sebaceous glands. The antigen epitope defined by MAb 12C3 was present on a glycoprotein with a molecular mass of 200 kDa and did not exhibit any cross-reactivity with other well-known tumor markers. These data suggest that MAb 12C3 may be a useful tool for the immunohistologic detection of early malignant changes in epithelial ovarian tumors. In addition, MAb 12C3 also may facilitate a differential diagnosis between benign and borderline malignancies.

Human aldolase B gene: characterization of the genomic aldolase B gene and analysis of sequences required for multiple polyadenylations.

The chromosomal gene encoding human aldolase B was isolated. The gene is composed of nine exons interrupted by eight introns and spans 15 kb, and a single copy of it occurs per haploid human genome. The initiation of transcription occurs at three different sites. Two minor sites, m1 and m2, start at 49 and 21 nucleotides, respectively, upstream from the major site, M. The gene also carries poly(A) addition signals at two different sites, thereby another two distinct mRNA species are produced. We examined the sequences required for mRNA 3'-end formation in this gene carrying multiple poly(A) addition sites. By constructing deletion mutants as to the region distal to the poly(A) addition site and then assaying through transfection into COS-1 cells, we demonstrated that 8 nucleotides distal to the site of poly(A) addition is sufficient for proximal polyadenylation, but is not sufficient for distal polyadenylation.

Copy-dependent and position-independent expression of rat aldolase A gene.

In order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1,2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.

Epstein-Barr virus genome-positive tubulointerstitial nephritis associated with immune complex-mediated glomerulonephritis in chronic active EB virus infection.

Renal involvement is rare in chronic active Epstein-Barr (EB) virus infection. We report a case of a 7-year-old girl with recurrent EB virus infection. She had fever, lymphadenopathy, hepatosplenomegaly, and persistently high titres of IgG to EB virus capsid antigen (VCA) and IgG to EB early antigen with low titres of IgM to VCA. She showed mild haematuria and proteinuria, but had no symptoms of renal failure. Renal biopsy revealed immune complex-mediated glomerulonephritis, which may have been due to a persistently high titre of antibody against EB virus. In addition, a peculiar form of tubulointerstitial nephritis was found. The morphology was characterized by a papillary infolding of the tubular epithelial cell layer into the tubular lumen. The interstitium was surrounded by the infolded epithelium and contained a large number of B-cell dominant lymphocytes. EBV-encoded RNA 1 (EBER-1) gene was detected in the nuclei of some tubuloepithelial cells by in situ hybridization and may have been associated with the pathogenesis of tubulointerstitial nephritis.

Diffuse mesangial sclerosis associated with Kawasaki disease: an analysis of alpha chains (alpha 1-alpha 6) of human type IV collagen in the renal basement membrane.

A case of diffuse mesangial sclerosis (DMS) associated with Kawasaki disease is reported. A previously healthy Japanese girl, aged 4 months, presented with clinical features of Kawasaki disease. At week 10 of the illness, she developed the nephrotic syndrome, which was refractory to steroid therapy. Renal biopsy demonstrated a diffuse mesangial proliferative glomerulonephritis with microcystic tubular dilatation and, ultrastructurally, marked thinning of the lamina densa in the glomerular basement membrane (GBM) and the tubular basement membrane (TBM) of the proximal tubule. She went into chronic renal failure and died at the age of 11 months. At autopsy, the kidney revealed DMS. Histologically, we found Finnish microcystic disease in its early stages in the biopsy. Using a newly developed monoclonal antibody, we analysed the alpha chains (alpha 1-alpha 6) of type IV collagen in the GBM and TBM. There was no defective constitution of alpha chains on the thin GBM, but the thin TBM of the microcystic proximal tubule showed a weak or discontinuous reactivity for alpha 1 and alpha 2 chains, suggesting faulty formation of the basement membrane. The sclerosing glomeruli of the DMS did not depend on collapse of the GBM, which was positive for alpha 3-alpha 5 chains, but mainly on the proliferation of mesangial matrix, which was positive for alpha 1 and alpha 2 chains.