Role of major histocompatibility complex class II in the development of autoimmune type 1 diabetes and thyroiditis in rats.

Although the MHC class II 'u' haplotype is strongly associated with type 1 diabetes (T1D) in rats, the role of MHC class II in the development of tissue-specific autoimmune diseases including T1D and autoimmune thyroiditis remains unclear. To clarify this, we produced a congenic strain carrying MHC class II 'a' and 'u' haplotypes on the Komeda diabetes-prone (KDP) genetic background. The u/u homozygous animals developed T1D similar to the original KDP rat; a/u heterozygous animals did develop T1D but with delayed onset and low frequency. In contrast, none of the a/a homozygous animals developed T1D; about half of the animals with a/u heterozygous or a/a homozygous genotypes showed autoimmune thyroiditis. To investigate the role of genetic background in the development of thyroiditis, we also produced a congenic strain carrying Cblb mutation of the KDP rat on the PVG.R23 genetic background (MHC class II 'a' haplotype). The congenic rats with homozygous Cblb mutation showed autoimmune thyroiditis without T1D and slight to severe alopecia, a clinical symptom of hypothyroidism such as Hashimoto's thyroiditis. These data indicate that MHC class II is involved in the tissue-specific development of autoimmune diseases, including T1D and thyroiditis.

[A case of bilateral renal cell carcinoma showing a rapid course of death in a long-term hemodialysis patient due to polycystic kidney].

A case of bilateral renal cell carcinoma in a 42-year-old polycystic kidney male is reported. He had been treated with hemodialysis for 22 years. An abnormal small mass was found in one of the left renal cystic lesions by screening ultrasonography and CT scan at the 19th year of the hemodialysis. Left radical nephrectomy was performed and the histological diagnosis was a renal cell carcinoma (RCC). There was no evidence of recurrence and metastasis, however, he presented with asymptomatic macrohematuria two years after the operation. CT scan demonstrated the rapidly progressing right renal tumor and multiple para-aortic lymph node swelling. Right nephrectomy and lymphadenectomy were performed and pathological examination showed the advanced RCC with multiple lymph node metastasis. Eleven months after the second operation followed by interferon therapy. he died of multiorgan metastasis of the RCC. This is the first bilateral RCC case in polycystic kidney patient treated with hemodialysis in Japan.

[Successful complete androgen blockade (CAB) therapy for prostatic cancer detected from multiple lung metastases: a case report].

Prostatic cancer is rarely diagnosed by detection of lung metastases. We report a case of prostatic cancer in a 73-year-old man detected by abnormalities in chest X-ray and serum prostate specific antigen (PSA) level. He was initially admitted to our hospital due to elevation of PSA level. On the first transperineal prostatic needle biopsy, prostatic cancer was not detected and he was followed. Seven months after the first biopsy, chest X-ray revealed multiple abnormal nodules in the lung fields bilaterally and PSA level was again elevated. A second prostatic biopsy and whole-body examination were performed, and he was diagnosed with moderately differentiated prostatic adenocarcinoma with multiple lung metastases. Complete androgen blockade therapy was performed immediately. Two months after the beginning of treatment, PSA level was normalized and the multiple lung metastases had completely disappeared. There has been no evidence of recurrence or PSA relapse 24 months after detection of the prostatic cancer. This is the 26th case of prostatic cancer diagnosed in Japan following detection of multiple lung metastases.

[Two cases of primary carcinoma developed in a vesical diverticulum treated with ileal neobladder for total bladder replacement].

Two cases of carcinoma developed in vesical diverticulum were encountered. Both these males, the first, 66-year-old and the second case, 68-year-old gentleman, visited our clinic with a complaint of asymptomatic gross hematuria. Total cystectomy was performed, and an ileal neobladder was constructed for total bladder replacement in both two cases. Pathological examination of the first case revealed transitional cell carcinoma, and the second case showed adenocarcinoma mixed with transitional cell carcinoma. Although primary carcinoma developed in vesical diverticulum was initially reported by Williams in 1883, adenocarcinoma is very rare. The diagnosis is often challenging and the prognosis is dire. Both our cases were successfully treated with total cystectomy and an ileal neobladder was constructed for total bladder replacement. Careful management and aggressive therapy for such patients may make even cure possible.

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis.

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18. 3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B. halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 sigma factors which belong to the extracytoplasmic function family, 10 are unique to B. halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.

Characterization and comparative study of the rrn operons of alkaliphilic Bacillus halodurans C-125.

The ribosomal RNA operons (rrn) of alkaliphilic Bacillus halodurans C-125 were characterized and compared with those of B. subtilis. We isolated clones containing rrn operons from a lambda phage library of the C-125 chromosome, and the complete nucleotide sequence of each was determined. Eight rrn operons were identified by PFGE analysis of the C-125 chromosome digested with I-CeuI. The transcriptional orientation of the rrn operons mapped on the chromosome by Southern hybridization analysis was the same as the direction of replication of the chromosome. These operons were designated as rrnA-H, starting from the oriC locus in clockwise rotation. Sequence and structural analyses of these operons suggested that six of the rrn operons in the C-125 chromosome, rrnA, rrnB, rrnC-rrnD, rrnE, and rrnH, correspond to rrnO, rrnA, rrnJ-rrnW, rrnI, and rrnD in B. subtilis, whereas the other rrn operons (rrnF and rrnG) were specifically observed in C-125. The rrn loci were positioned from 0 degrees to 90 degrees on the physical map, with the oriC locus assigned the position zero degrees. Two ORFs annotated as tnpA and ykfC, whose gene products are likely to act as transposases, were found downstream of these six operons. Comparative analysis of the 16S-23S and 23S-5S ITS (internally transcribed sequence) regions of B. halodurans C-125 and those of B. subtilis revealed that the ITS regions in C-125 were much longer than those in B. subtilis. There was no substantial difference in the length of potential promoter sequences in B. halodurans and B. subtilis.

Genetic analysis of the chromosome of alkaliphilic Bacillus halodurans C-125.

Seventeen Sse8387I linking clones isolated from the chromosome of Bacillus halodurans C-125 for the purpose of constructing a physical map were sequenced and analyzed by comparison with the BSORF database and the nonredundant protein databank. The orientations of Sse8387I or AscI linking clones serving to join adjacent fragments were determined by southern blot analysis using specific DNA probes. One-third of the open reading frames (ORFs) identified in the Sse8387I linking clones showed no significant similarity to any protein so far reported. The ORFs showing significant similarities to those of Bacillus subtilis were mapped in the chromosome of strain C-125, and the locations of the putative genes on the map were not well conserved between B. halodurans C-125 and B. subtilis.

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.

An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125.

Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region.

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125.

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15-20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a lambda phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis.

New Method of Screening for Pressure-sensitive Mutants at High Hydrostatic Pressure.

We have developed an efficient method of screening to detect pressure-sensitive mutants of barophilic or barotolerant bacteria using conventional agar medium plates. By this new method, 75 colonies can be screened per plate under high hydrostatic pressure.

Photobacterium profundum sp. nov., a new, moderately barophilic bacterial species isolated from a deep-sea sediment.

A novel, moderately barophilic bacterium was isolated from a sediment sample obtained from the Ryukyu Trench, at a depth of 5110 m. The isolate, designated strain DSJ4, is a Gram-negative rod capable of growth between 4 degrees C and 18 degrees C under atmospheric pressure, with optimum growth displayed at 10 degrees C, and capable of growth at pressures between 0.1 MPa and 70 MPa at 10 degrees C, with optimum growth displayed at 10 MPa. Strain DSJ4 is a moderately barophilic bacterium, and shows no significant change in growth at pressures up to 50 MPa. Phylogenetic analysis of the 16S rRNA sequence of strain DSJ4 places this strain within the Photobacterium subgroup of the family Vibrionaceae, closely related to the strain SS9 that was independently isolated from the Sulu Trough. The temperature and pressure ranges for growth, cellular fatty acid composition, and assorted physiological and biochemical characteristics indicate that these strains differ from other Photobacterium species. Furthermore, both SS9 and DSJ4 displayed a low level of DNA similarity to other Photobacterium type strains. Based on these differences, these strains are proposed to represent a new deep-sea-type species. The name Photobacterium profundum (JCM10084) is proposed.

A denitrifying bacterium from the deep sea at 11,000-m depth.

The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30 degrees C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4 degrees C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies.

[Endothelin levels after extracorporeal shock waves, effects of renal function and blood pressure in rats].

BACKGROUND: Endothelin is a recently discovered peptide with strong vasoconstrictive effects which is of potential relevance in the development of renal hypertension and renal failure. It is secreted by endothelial cells in response to ischemia, endothelial lesions and inflammatory or traumatic processes. Endothelin level after extracorporeal shock waves, effects of renal function and blood pressure were examined in rats. METHODS: Changes of plasma endothelin-1 (ET-1) levels after extracorporal shock waves were studied by applying 500 shock waves to both kidneys in Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Plasma ET-1 was measured before shock wave exposure and every thirty minutes up until 2 hours, then at 12 hours and 24 hours after shock wave exposure by specific sandwich enzyme immunoassay (EIA). Findings were then compared with those from the control group. Mean arterial pressure (MAP), glomerular filtration rate (GFR) and renal plasma flow (RPF) in rats were measured before shock wave exposure and every thirty minutes up until 2 hours after shock wave exposure, moreover made evaluation when ET-1 specific antibodies were administered intravenously into WKY and SHR. RESULTS: Plasma ET-1 was significantly increased in WKY (n = 6) and SHR (n = 7) after shock wave exposure as compared with that in the control group (p < 0.05). In SHR (n = 6), GFR temporarily decreased at 60 min but RPF remarkably decreased and renal vascular resistance (RVR) increased at 30 approximately 120 min after shock wave exposure (p < 0.05). However, infusion of ET-1 specific antibodies into SHR curbed change in those parameters, increased GFR and RPF at 60 approximately 120 min after shock wave exposure (p < 0.05). In WKY (n = 5), RPF temporarily decreased and RVR slightly increased, infusion of ET-1 specific antibodies curbed change in those parameters. MAP in SHR slightly increased at 30 min after shock wave exposure, but there was no significant change in WKY. CONCLUSION: These results suggest that plasma ET-1 levels elevated in both strains and ET-1 influenced renal function after extracorporeal shock waves, and that SHR is more sensitive to ET-1 in comparison with WKY.

[Changes of plasma endothelin level in the early post-renal transplantation period].

Endothelin (ET) has been suggested to be involved in acute graft rejection of kidney transplantation and cyclosporin A (CsA) nephrotoxicity. For clarification of the pathophysiological role of ET in the early post-transplantation period, plasma endothelin-1 (ET-1) was measured by specific radioimmunoassay in renal transplant recipients, patients on maintenance hemodialysis (HD) and healthy volunteers. Twelve transplant recipients were used in this study, 8 of whom were living related subjects and 4 cadaver. Plasma ET-1 and graft function were measured each day, from 1 day prior and 7 days following transplantation and every week up to 5 weeks postoperatively. Plasma ET was measured in 20 other transplant recipients with stable function (serum creatinine < or = 1.8 mg/dl), 20 maintenance HD patients with no residual renal function and 6 healthy volunteers. Mean plasma ET-1 was 13.0 +/- 4.5 pg/ml in 20 recipients with stable graft function, 21.7 +/- 6.5 in 20 HD patients and 1.5 +/- 0.4 in healthy volunteers. These differences are statistically significant (p < 0.02). Plasma ET-1 showed significant decrease from 21.8 +/- 7.2 pg/ml prior to transplantation to 12.8 +/- 4.0 when urinary output reached more than 1000 ml in living and cadaveric transplantation subjects. All three acute vascular rejections clearly indicated histologically increased plasma ET-1 accompanied by an increase in serum creatinine. A significant positive liner correlation was noted between plasma ET-1 and serum creatinine during the first week following living transplantation. Two patients with clinically and histologically suspected CsA nephrotoxicity showed transient increase in plasma ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of malignant mesothelioma of the tunica vaginalis testis].

A case of malignant mesothelioma of tunica vaginalis testis experienced in an 83-year-old male who presented with a complaint of left intrascrotal pain is reported. The tentative diagnosis was acute epididymitis. Left epididymectomy was performed following the unsuccessful treatment with antibiotics. The histology of the surgical specimen revealed malignant mesothelioma. Radical excision of the left spermatic cord and scrotal content indicated locally advanced disease with positive surgical margin. Metastatic workup, including abdominal and chest computerized tomography scan, magnetic resonance imaging and bone scan demonstrated presence of metastasis to the paraaortic lymph nodes and the lumber spine. The patient is being followed conservatively in consideration of his age and advanced stage of the disease.

Generation of fractal patterns for probing the visual memory.

The effective use of computer-generated pictures as a trial-unique probe for studying the visual memory is described. The shape of the pattern is determined by means of a fractal algorithm with pseudorandom parameters. This method enables us to easily obtain thousands of moderately complex and sufficiently diversified pictures in series from a given number which serves as the seed of a pseudorandom number generator. We can thereby create a new and unique set of pictures if a new seed is given, as well as retrieve exactly the same pictures in the same sequence as when the original seed is given. These properties eliminate the demand for the massive memory space in a computer otherwise needed to store the entire set of stimulus pictures.

Alternative splicing of hepatitis B virus RNAs in HepG2 cells transfected with the viral DNA.

We identified a novel spliced RNA of 2.6 kb from a human hepatoma cell line HepG2 transfected with the hepatitis B virus (HBV) genome. The splicing acceptor site of the novel 2.6-kb RNA (position 489) was shown to be common to that of the previously described 2.1-kb spliced RNA which codes for an altered core antigen lacking the carboxy-terminal amino acid, cysteine. However, the donor site of the 2.6-kb RNA is different from any of the spliced RNA reported and located at 538 nucleotides (nt) downstream of the donor site of the 2.1-kb RNA. Introduction of single-base change mutations in the consensus sequence of the donor site of the 2.1-kb RNA maintained the splicing by using the cryptic donor site. The amount of the 2.6-kb spliced RNA was unchanged by these mutations. These results suggest independent regulations for the synthesis of the 2.1- and 2.6-kb spliced RNAs.

Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome.

HepG2 cells, known to support the replication and virion formation of hepatitis B virus (HBV), were transfected with a cosmid constructed to contain 12 tandem head-to-tail repeats of the HBV genome for effective HBV genome expression. We detected previously identified RNAs of 3.3, 2.3, and 2.0 kilobases (kb) that code for core antigen, large surface antigen, and middle/major surface antigen, respectively. We also detected four additional RNAs of 2.1, 1.7, 1.1, and 0.7 kb [the lengths exclude the poly(A) tail]. S1 mapping and nucleotide sequencing data showed that the 2.1-kb RNA is a spliced RNA whose 5' and 3' ends are identical to those of the 3.3-kb RNA. The results suggest that the 2.1-kb RNA codes for an altered core antigen lacking the last amino acid, cysteine, and that expression of the 3.3-kb pregenomic RNA is regulated, at least in part, by splicing. The map positions of the 1.7- and 1.1-kb RNAs suggest that they code for the carboxyl-terminal portions of the putative polymerase, whereas the 0.7-kb RNA codes for the X protein.