Cloning of the peroxiredoxin gene family in rats and characterization of the fourth member.

Peroxiredoxin (PRx) exhibits thioredoxin-dependent peroxidase activity and constitutes a family of proteins. Four members of genes from rat tissues were isolated by PCR using degenerated primers based on the sequences which encode a pair of highly conserved Cys-containing domains, and were then cloned to full-length cDNAs. These included two genes which have previously been isolated in rats, PRx I and PRx II, and two rat homologues of PRx III and PRx IV. We showed, for the first time, the simultaneous expression of all four genes in various rat tissues by Northern blotting. Since a discrepancy exists regarding cellular distribution, we further characterized PRx IV by expressing it in COS-1 cells. This clearly demonstrates that PRx IV is a secretory form and functions within the extracellular space.

Visible integration of the adenosine deaminase (ADA) gene into the recipient genome after gene therapy.

Gene therapy for patients with adenosine deaminase (ADA) deficiency has become practical in the 1990s, and the exogenous gene has been reported to survive for several years in the recipient genome. To evaluate the integration efficiency of the ADA gene (ADA) into peripheral blood lymphocytes (PBL) of a patient with ADA deficiency who is receiving gene therapy, we performed two-color interphase fluorescence in situ hybridization (FISH) analysis by using digoxigenin-labeled ADA-cDNA and the biotin-labeled lambda-genomic ADA clone as probes. After each of 9 sequential series of gene therapy, interphase nuclei of 100 mononuclear cells from the patient were analyzed, and those of a LASN-producing cell line were used as a control. FISH signals were detected with rhodamine and FITC for the cDNA and the genomic DNA, respectively. The number of PBL giving a transgene signal grew after the sequential gene therapies, and the proportion of signal-positive cells reached about 10%. Our results indicate that the two-color FISH system can be used as a potential aid to monitor the efficiency of the ADA gene therapy.

Diagnosis of four chromosome abnormalities of unknown origin by chromosome microdissection and subsequent reverse and forward painting.

A molecular cytogenetic method consisting of chromosome microdissection and subsequent reverse/forward chromosome painting is a powerful tool to identify chromosome abnormalities of unknown origin. We present 4 cases of chromosome structural abnormalities whose origins were ascertained by this method. In one MCA/MR patient with an add(5q)chromosome, fluorescence in situ hybridization (FISH), using probes generated from a microdissected additional segment of the add(5q) chromosome and then from a distal region of normal chromosome 5, confirmed that the patient had a tandem duplication for a 5q35-qter segment. Similarly, we ascertained that an additional segment of an add(3p) chromosome in another MCA/MR patient had been derived from a 7q32-qter segment. In a woman with a history of successive spontaneous abortions and with a minute marker chromosome, painting using microdissected probes from the whole marker chromosome revealed that it was i(15)(p10) or psu dic(15;15)(q11;q11). Likewise, a marker observed in a fetus was a ring chromosome derived from the paracentromeric region of chromosome 19. We emphasize the value of the microdissection-based chromosome painting method in the identification of unknown chromosomes, especially for marker chromosomes. The method may contribute to a collection of data among patients with similar or identical chromosome abnormalities, which may lead to a better clinical syndrome delineation.

Resonance energy transfer study of peptide-lipid complexes.

Resonance energy transfer involving tryptophan as a donor and anthrylvinyl-labeled phosphatidylcholine (AV-PC), 3-methoxybenzanthrone (MBA) and 8-anilino-1-naphthalene sulfonic acid (ANS) as acceptors has been examined to obtain information on the structure of peptide-lipid systems consisting of 18A or Ac-18A-NH(2) peptides and large unilamellar phosphatidylcholine vesicles. The lower and upper limits for the tryptophan distance from the bilayer midplane have been assessed in terms of the models of energy transfer in two-dimensional systems, taking into account orientational effects. Evidence for the existence of preferential orientations of Ac-18A-NH(2) with respect to the lipid-water interface has been obtained.

Effects of the acyl chain composition of phosphatidylcholines on the stability of freeze-dried small liposomes in the presence of maltose.

The effects of the acyl chain composition of phosphatidylcholines (PCs) on the stability of small unilamellar vesicles during freeze-drying and rehydration in the presence of maltose were studied by monitoring the retention of a trapped marker, calcein, in the internal liposome compartment. In dipalmitoyl PC, beta-oleoyl-gamma-palmitoyl-PC and egg yolk PC liposomes, good or fair retentions (>50%) were observed in the presence of maltose, but maltose was ineffective in preserving retention in the dioleoyl PC (DOPC) liposomes (<10%). The extremely low retention in the DOPC liposome was ascribed to neither a formation of the inverted hexagonal phase of the liposomal membrane nor the fusion/aggregation of the liposomes in the drying-rehydration process. Differential scanning calorimetry measurements suggested that interactions of maltose with PC headgroups were essential to stabilizing the dry liposomes. These interactions were significant in the saturated or mixed chain liposomes but were markedly reduced in the DOPC liposomes.

Anterior communicating artery aneurysm in early childhood. Report of a case.

A case of an aneurysm of the anterior communicating artery in a 13-month-old child is presented. Sixty-six cases of cerebral saccular aneurysm in children under the age of 2 years found in the literature were analyzed. The characteristics that define this group of patients are a high frequency of large or giant aneurysms, a large proportion of aneurysms of the middle cerebral artery and the posterior circulation, and the frequent location at the peripheral site. Only three cases of anterior communicating artery aneurysm were reported in the literature. The authors discuss these characteristics on the basis of development of fetal cerebral vessels.

Two nonsense mutations of PAX6 in two Japanese aniridia families: case report and review of the literature.

PURPOSE: To identify PAX6 mutations in patients from four Japanese families with aniridia. METHODS: Polymerase chain reaction (PCR)-single stand conformational polymorphism (SSCP) analysis (SSCA) was performed in probands of the families, and restriction analysis using MaeIII or AvaI was carried out in other affected family members. RESULTS: PCR-SSCA demonstrated in the proband from one family an extra-band in the PCR product for PAX6 exon 8. Base sequence analysis revealed that the patient is a heterozygote for a C to T transition mutation at codon 203. DNAs from the patient and another affected member in the same family were cut with MaeIII into two fragments, while non-affected members in the family showed only one MaeIII fragment, the result confirmed the mutation. In another family, PCR-SSCA revealed an extra-band in the PCR product for exon 9. Sequencing detected a C-->T substitution at codon 240 in the patient, the mutation resulted in loss of an AvaI site. AvaI cleavage analysis confirmed the mutation in the patient. The two transition mutations observed in the two families also predict the conversion of arginine to a stop codon (R203X and R240X, respectively) around the homeodomain (HD), leading to the truncation of the PAX6 protein within its glycine-rich region. No abnormal SSCP bands or abnormal restriction fragments were detected in patients from the other two families. CONCLUSIONS: The two mutations sites identified in the two families, one at codon 203 and the other at codon 240, are those most frequently observed among 118 previously reported PAX6 mutations. This indicates that the two mutations are two hot-spots in the gene.

[Clinical efficacy of fosfomycin in combination with sulbactam/cefoperazone in the treatment of severe infections complicated to blood dyscrasia. Working Group of Kanto Combination Therapy for FOM + SBT/CPZ].

In the treatment of severe infections complicated to blood dyscrasia, the efficacy and usefulness of fosfomycin (FOM) in combination with sulbactam (SBT)/cefoperazone (CPZ) were compared between patients receiving FOM in the first followed by SBT/CPZ (Group A) and those receiving both drugs simultaneously (Group B). The following results were obtained. 1. The efficacy rate was 56.3% for Group A and 47.9% for Group B, with no significant difference. 2. The efficacy for patients suspected of the presence of septicemia, the efficacy rate was 57.9% for Group A and 54.3% for Group B, with no significant difference. 3. As for underlying disease, patients with acute myelogenous leukemia were most prevailing. In these patients, the efficacy rate was 57.1% for Group A and 27.3% for Group B, with no statistically significant difference. However, the efficacy rate tended to be higher in Group A. 4. The administration of antibiotics was effective to restore the neutrophil count to 501/microliters or higher in 77.8% and 45.5% of the cases for Groups A and B, respectively, with significantly higher efficacy for Group A. 5. In the safety evaluation a total of 115 cases were included. Side effects and laboratory abnormalities were seen in 3 cases each, but none of them were serious in degree. From these results, it was confirmed that the combination therapy consisting of administration of FOM followed by SBT/CPZ with some interval is effective for severe infections complicated to blood dyscrasia.

[Spontaneous spinal epidural hematoma diagnosed by MRI: a case report].

Appropriate diagnostic procedure for spinal epidural hematomas has not been established yet. The authors reported a case of spontaneous epidural hematomas at the thoracic level, in which correct diagnosis was made with MRI and good results were obtained by surgery. A 63-year-old female experienced a severe back pain which appeared suddenly during a walk and was followed by motor weakness in both legs deteriorating quickly to paraplegia. The patient had no history of hypertension, trauma or bleeding tendency. The laboratory data were normal. On admission, neurological examination revealed flaccid paraplegia, total sensory loss below the level of Th 6 and urinary and fecal incontinence. Myelograms showed incomplete block at the Th 6 level and postmyelographic CT scan showed an isodense mass, which was suspected to be an epidural tumor located behind the spinal cord. Emergent MRI confirmed an epidural hematoma as a high intensity area extending from Th 3 through Th 11. Sixty-five hours after onset, laminectomy of Th 4 through Th 11 and the evacuation of epidural hematoma were performed without identification of the origin of the bleeding. Neither vascular malformation nor tumor was recognized during operation. Neither was it noticed on histological examination. The patient made favorable progress after the surgery. During the first two weeks in the postoperative period, she regained muscle strength enough to do standing exercise, and satisfactory improvement was made in sensory function including urination and defecation. We emphasize that MRI is indispensable to make a differential diagnosis of thoracic lesions. In the reported case, a correct diagnosis was made with MRI, and an extremely good result was obtained by an emergency operation.

[Inflammatory granulomas extending from the sphenoid sinus to the cavernous sinus: report of three cases].

We treated three cases of inflammatory granulomas extending from the sphenoid sinus to the cavernous sinus. Case 1 was that of a 36-year-old male with diplopia and right ptosis. Case 2 was that of a 40-year-old male with frontal headache. Case 3 was that of a 70-year-old female with left impaired vision and frontal headache. In the first case, MRI demonstrated a mass lesion extending from the right half of the sphenoid sinus to the cavernous sinus and retropharyngeal space. In the second case the granuloma extended from the right cavernous sinus to the right retroorbital space. In the last case, MRI demonstrated diffuse Gd-DTPA enhancement of the left cavernous sinus and the left half of the sphenoid sinus. In all cases an operation was performed using the sublabial rhinoseptal approach, and the tumor in the sphenoid sinus was removed. Histological examination revealed an inflammatory granuloma in all 3 cases. In the first case the clinical symptoms improved following administration of glucocorticoids. In the second case the mass in the cavernous sinus decreased in size postoperatively. In the last case, the clinical symptoms gradually improved with administration of antibiotics after surgery. Granuloma of the cavernous sinus is difficult to diagnose, but when a similar pathological lesion coexists in the sphenoid sinus, a definitive diagnosis can be easily made via the sublabial rhinoseptal approach.

[A case report of dural sinus thrombosis: direct thrombolytic therapy using endovascular surgery].

Dural sinus thrombosis, a relatively rare disease, is difficult to diagnose because of variable symptomatic manifestations. We successfully treated a case of dural sinus thrombosis by direct thrombolysis using an endovascular technique in combination with postoperative anticoagulant therapy. The patient, a 19-year-old female, developed a headache affecting her whole head on December 13, 1993. She was admitted to our hospital the next day. Neurological examination upon admission revealed no neurological abnormalities, nor was there any abnormality in CT scan, either plain or enhanced, taken on the day of admission. The patient's consciousness deteriorated in the early morning of December 22. MRI and cerebral angiography revealed thrombi from the confluence of the sinuses to the right transverse and sigmoid sinus, with disturbed circulation through deep cerebral veins. Systemic thrombolytics, steroid and mannitol were started, but, on the next day, the third ventricle was compressed by bilateral swelling of the basal ganglia, with hydrocephalus. Since her consciousness deteriorated further despite ventricular drainage and barbiturate therapy, direct thrombolytic therapy was performed on December 25. A catheter was placed in the superior sagittal sinus, and 600,000 units of urokinase was locally injected, followed by postoperative anticoagulant therapy. The patient's condition improved rapidly. On CT scan, the bilateral swelling of the basal ganglia disappeared along with the hydrocephalus. At about 1 month after endovascular surgery, MRI and cerebral angiography revealed recanalization of the deep cerebral veins, straight sinus and confluence of sinuses with improved opacification of the left transverse sinus, although the right transverse sinus was found to be re-occluded.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of metastatic spinal tumor from renal cancer reduced by interferon-alpha].

A case of metastatic spinal tumor from renal cancer reduced by interferon-alpha is reported. A 54-year-old man was admitted to Tane General Hospital on April 12, 1990, with the complaint of difficulty in walking. Abdominal CT scan revealed right renal carcinoma, and MRI demonstrated metastatic spinal tumor at Th6, which was mainly occupying the entire lamina, spinous process and right pedicle of Th6. Nephrectomy was performed and, instead of operating on the spinal lesion, the patient was given follow-up treatment with administration of interferon-alpha (5 million units twice a week) and steroid. 1 year and 6 months later the patient's walking ability began to deteriorate and paraparesis progressed, although the size of the metastatic spinal tumor had gradually reduced. This was thought to be because of atrophy of the legs due to prolonged bed rest and steroid administration. Laminectomy with total removal of the tumor was performed on January 16, 1992. In the 12 months postoperatively, there has been neither recurrence nor remote metastasis seemingly due to the administration of interferon-alpha. This appears to be the first report of metastatic spinal tumor from renal cancer reduced by interferon-alpha.

[Characterization of peripheral blood natural killer (NK) cells in two patients with CD16+ CD56- NK cell-lineage granular lymphocyte-proliferative disorder].

In normal peripheral blood natural killer (NK) cells, the subset of CD16+ CD56+ cells is predominant, and that of CD16+ CD56- cells is rarely present. Because we have found the expansion of CD16+ CD56- NK cells in the peripheral blood of two patients with NK cell-lineage granular lymphocyte-proliferative disorders (NK-GLPD), the clinical findings and cellular characteristics of these patients were compared with those of CD16+ CD56+ NK-GLPD patients. Although CD16+ CD56- and CD16+ CD56+ NK-GLPD cells were morphologically different, clinical findings and courses, and NK activity did not differ significantly. Because strong NK activity was demonstrated in CD16+ CD56- NK-GLPD cells, the CD56 antigen, one of the adhesion molecules, did not seem to play a major role in NK cell-mediated cytotoxicity. The CD56 antigen is known to be more strongly expressed by immature NK cells than by mature NK cells. However, because interleukin 2-activated CD16+ CD56- NK-GLPD cells rapidly expressed the CD56 antigen, the degree of CD56 antigen expression did not always correlate with the maturity of NK cells.

Direct measurement of CD34+ blood stem cell absolute counts by flow cytometry.

For the collection of adequate numbers of peripheral blood stem cells (PBSC) for PBSC transplantation, an accurate quantification of circulating CD34+ stem cells is required for deciding the optimal time of the collection. To enumerate peripheral blood (PB) CD34+ stem cells, the percentage of CD34+ cells in the gated PB mononuclear cells should be multiplied by the percentage of the gated mononuclear cells among white blood cells (WBC) and by the total WBC count. Accordingly, a minor difference in the measured percentage of the CD34+ cells can lead to a major difference in the PB CD34+ cell concentration. In the present study, we measured the concentration of PB CD34+ stem cells with a flow cytometer designed to provide direct absolute counts of cell subsets from a single instrument. Whole blood was stained with a phycoerythrin-conjugated anti-CD34 monoclonal antibody, and, after the lysis of red blood cells, CD34+ cells were counted in a fraction of the lymphocyte and monocyte gate. The accuracy of our method was demonstrated in an experiment in which various dilutions of known numbers of CD34+ leukemic cells were mixed with normal blood; the predicted value of the CD34+ cell count was observed. The concentration of CD34+ cells in leukapheresis products was measured both by our direct assay and an indirect assay that calculates the number from the percentage of CD34+ cells in mononuclear cells, and our assay was shown to produce less variation. Further, our assay showed a significant correlation between the concentration of mobilized CD34+ cells in the PB and the number of harvested CD34+ cells in leukapheresis. These findings indicate that the monitoring of the concentration of PB CD34+ cells by the present method can be used to predict the number of stem cells collected in leukapheresis. This procedure is easy to perform and can be applied to daily monitoring to decide the appropriate timing for harvest of mobilized stem cells.

Differentiation stage of natural killer cell-lineage lymphoproliferative disorders based on phenotypic analysis.

In the normal developmental pathway of natural killer (NK) cells, pre-NK cells express CD161, immature NK cells express CD161 and CD56, and mature NK cells express CD161, CD56 and CD94. To identify the normal counterpart of NK cells from which neoplastic cells originate, surface antigens were analysed. Blastic NK-cell lymphoma/leukaemia lacked CD94 and CD161 but had CD56. Aggressive NK-cell leukaemia/lymphoma and nasal NK-cell lymphoma, although morphologically immature, expressed both CD56 and CD94 and strong NK activity. Cells from chronic NK lymphocytosis expressed CD56 and CD94.

The human HCLS1 gene maps to chromosome 3q13 by fluorescence in situ hybridization.

The human HS1 gene (HCLS1, hematopoietic cell-specific Lyn substrate 1) expressed in human hematopoietic cells encodes a major substrate of protein-tyrosine kinase, p75HS1. This intracellular protein is involved in the signal transduction pathways that initiate at the antigen receptors of both B and T lymphocytes. Fluorescence in situ hybridization using a 2.0-kb cDNA and an 8.0-kb genomic DNA clone of HCLS1 as probes revealed that the gene maps to 3q13.

Cancer-associated alternative usage of multiple promoters of human GalCer sulfotransferase gene.

The galactosylceramide sulfotransferase (cerebroside sulfotransferase, CST) (EC 2.8.2.11) gene is highly expressed in human renal cancer cells. To elucidate the regulatory mechanism of its gene expression, we have determined the genomic organization of the human CST gene. The gene comprises at least four exons and spans about 20 kb. The coding region is located in exons 3 and 4. To determine the transcription initiation sites, 5'-rapid amplification of cDNA ends analysis was performed using mRNA obtained from four human renal cancer cell lines, SMKT-R1-R4, and normal human renal proximal tubular cells. We found four transcription initiation sites and alternative usage of six exons corresponding to the 5'-untranslated region in cancer cells. On the other hand, the only transcript beginning at exon 1a was observed in normal cells. Using reverse transcriptase-PCR analysis, we confirmed that all of the exons 1a-d, especially exons 1c and 1d, are used as a transcription initiation site in cancer cells, whereas only exons 1a and 1b, mostly 1a, are utilized in normal cells. Analyzing the protein production from the mRNA variants with different 5'-UTRs, we found that all the transcripts examined produced the identical proteins. These observations suggest that the aberrant usage of transcription initiation sites flanked with promoters/enhancers is involved in the cancer-associated expression of the CST gene. Furthermore, this gene was assigned to human chromosome 22q12 by means of fluorescence in situ hybridization.

P-glycoprotein expression on normal and abnormally expanded natural killer cells and inhibition of P-glycoprotein function by cyclosporin A and its analogue, PSC833.

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by the MDR1 gene, has been found to be expressed in many normal bone marrow and peripheral blood cells. Among normal leukocytes, CD3(-)CD16(+) or CD3(-)CD56(+) lymphocytes, ie, natural killer (NK) cells, express relatively high levels of P-gp, but little is known about P-gp in abnormally expanded NK cells. In this study, we examined the expression and activity of P-gp on NK cells derived from three normal donors, six patients with indolent NK cell-lineage granular lymphocyte-proliferative disorder (NK-GLPD), three patients with aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell lymphoma), and two NK cell lines. By flow cytometric analysis using the monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp expression and the efflux of Rh123 were found in all NK samples except one NK cell line. The Rh123 efflux of NK cells was inhibited by cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK tumor cells were less inhibited than were the other NK cells. The percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% +/- 0. 9%, 93.4% +/- 3.1% and 36.9% +/- 11.7%, respectively, by 1 micromol/L CsA, and 80.2% +/- 3.6%, 91.7% +/- 2.6% and 32.7% +/- 10. 1%, respectively, by 1 micromol/L PSC833. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the low inhibitory effect of P-gp modulators in aggressive NK cell tumors did not correlate to the expression level of MDR1 gene, multidrug resistance-associated protein gene, or human canalicular multispecific organic anion transporter gene. This phenomenon could be related to the presence of other transporters or to unknown cellular or membrane changes. Some patients with NK cell tumors have been reported to show a highly aggressive clinical course and to be refractory to chemotherapy, and this could be related to the expression of P-gp on NK cells. Our results suggest that, although the inhibitors for P-gp have been used in combination with chemotherapy in some hematologic tumors, these inhibitors may be less effective against aggressive NK cell tumors.