Aquaporin and aquaglyceroporin in silkworms, differently expressed in the hindgut and midgut of Bombyx mori.

Two cDNAs similar to aquaporins (AQPs) from other insect species were identified and characterized from the silkworm larva, Bombyx mori. The first cDNA (AQP-Bom1) cloned from the anterior silk gland encodes a 25 900 Da protein similar to insect AQPs isolated from several liquid-feeding insects. The second cDNA (AQP-Bom2) cloned from the posterior midgut encodes a 27 694 Da protein. Northern blot analysis has revealed that the AQP-Bom1 mRNA (2.3 kb) is expressed predominantly in the hindgut (colon and rectum), and moderately or minimally in the silk gland, midgut and Malpighian tubules, while the AQP-Bom2 mRNA (1.3 kb) is mainly expressed in the posterior midgut and Malpighian tubules. Functional analysis in Xenopus oocytes microinjected with the cRNA of these AQPs revealed that the AQP-Bom1 mRNA encodes a water-specific aquaporin, likely involved in the water retrieval function of the hindgut, while the AQP-Bom2 mRNA encodes an aquaglyceroporin, increasing glycerol and urea uptake.

Transportin-SR, a nuclear import receptor for SR proteins.

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain- bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin beta/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.

Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase-M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1-A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.

Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.

Nonsense-mediated messenger RNA (mRNA) decay, or NMD, is a critical process of selective degradation of mRNAs that contain premature stop codons. NMD depends on both pre-mRNA splicing and translation, and it requires recognition of the position of stop codons relative to exon-exon junctions. A key factor in NMD is hUpf3, a mostly nuclear protein that shuttles between the nucleus and cytoplasm and interacts specifically with spliced mRNAs. We found that hUpf3 interacts with Y14, a component of post-splicing mRNA-protein (mRNP) complexes, and that hUpf3 is enriched in Y14-containing mRNP complexes. The mRNA export factors Aly/REF and TAP are also associated with nuclear hUpf3, indicating that hUpf3 is in mRNP complexes that are poised for nuclear export. Like Y14 and Aly/REF, hUpf3 binds to spliced mRNAs specifically ( approximately 20 nucleotides) upstream of exon-exon junctions. The splicing-dependent binding of hUpf3 to mRNAs before export, as part of the complex that assembles near exon-exon junctions, allows it to serve as a link between splicing and NMD in the cytoplasm.

Population dynamics of anaerobic microbial consortia in thermophilic methanogenic sludge treating paper-containing solid waste.

To maintain a stable thermophilic (55 degrees C) anaerobic digestion treating toilet paper-containing garbage, it is necessary to operate the digester at long hydraulic retention time (HRT) and low organic loading rate (OLR). Critical conditions of the digestion were investigated by operating the digester at HRT 23 days and OLR 3.4 gCOD(Cr)/L/d (R1) or HRT 14 days and OLR 5.6 gCOD/Cr)/L/d (R2) separately. Characteristics of methanogenesis of the two digesters were examined by measuring gas generation volume and volatile fatty acids (VFA) concentration, and the populations of four anaerobic acidogens and three methanogens were analyzed using quantitative PCR method. In digester R1, methanogenic activity was unstable but it could be recovered by stopping feeding as though VFA accumulation occurred. The population of acidogens and two methanogens were maintained at 10(11) - 10(13) copies/L, however, the population of Methanoculleus could not be recovered after methanogenesis recovering. In digester R2, the period of methanogenesis was significantly shorter than that in digester R1. Both the acidogens and the methanogens could not be maintained at a stable concentration. It is suggested that the critical HRT to sustain the population of acidogens in this process should be longer than 14 days and for all kinds of methanogens, HRT should be longer than 23 days.

Molecular genetic and immunohistochemical study of autosomal recessive Alport's syndrome.

A DNA analysis of autosomal type IV collagen alpha3 and alpha4 chain genes (COL4A3 and COL4A4) and an immunohistochemical study of type IV collagen alpha1 to alpha6 chains were performed in an inbred family with autosomal recessive Alport's syndrome (AS). A linkage study using polymorphic markers around the COL4A3/COL4A4 genes clearly differentiated the affected patients from healthy individuals. These patients were homozygous for all markers analyzed, whereas their parents were heterozygotes. Because of the large size of both the genes and the heterogeneous range of the mutations of these genes, linkage analysis by using highly polymorphic markers is still the method of choice in genetic counseling for autosomal recessive AS, as well as for the X-linked form. Although the distribution of alpha1 and alpha2 chains in the index patient and her affected sister were normal, the alpha3 and alpha4 chains were completely defective in the renal basement membrane (BM). The alpha5 chain could be found in Bowman's capsular basement membrane (BCBM) but not in the glomerular basement membrane (GBM). In addition, our study showed, for the first time, that the alpha6 chain in BCBM is spared in this form of AS. This abnormal pattern of type IV collagen could be a useful tool for differentiation of the autosomal recessive type from the X-linked type of AS.

Testing for rubella-specific IgG antibody in urine.

BACKGROUND: In Japan rubella vaccination is generally done once during a lifetime, and the vaccination rate decreased after a revised vaccination law in 1995. History of rubella or vaccination may still be unreliable. Testing for rubella antibody is significant to prevent the occurrence of congenital rubella syndrome. However, the collection of blood samples to detect antibodies from young children is invasive and difficult. METHODS: For this study we obtained 853 matched serum and urine samples from 904 healthy students 10 or 14 years of age in the Ibara and Yoshii districts of Okayama, Japan, for a comparison of antibodies for rubella in the matched samples. The serum and urine antibodies were measured with hemagglutination-inhibition and enzyme-linked immunosorbent assays, respectively, and with our urine-based antibody test. RESULTS: The sensitivity, specificity and concordance rates of this urine-based antibody test were 96, 99 and 97% based on the serum antibody results of both assays. The coefficiency was 0.627 between the titers of the urinary and serum antibodies by the enzyme-linked immunosorbent assay. The urinary antibodies were stable for at least 5 months at 4 degrees C and 25 degrees C. CONCLUSIONS: Urine-based assay methods are helpful not only because they avoid the invasive approach of venipuncture but also because unprocessed urine specimens can be used and urinary antibody is stable for a long period. Therefore this test is suitable for screening. In addition protective amounts of rubella antibody in blood can be reliably assessed by means of urine samples.

A case of hemolytic uremic syndrome improved with nitric oxide.

Hemolytic uremic syndrome (HUS) after transplantation is difficult to treat, and there is no consensus regarding optimal mode of treatment. We attached transdermal isosorbide tape as a nitric oxide (NO) donor to patients with HUS after bone marrow transplantation (BMT). This was very effective in ameliorating the hemolysis and increasing platelet numbers. We report here the successful use of an isosorbide in a patient with HUS after transplantation. Bone Marrow Transplantation (2000) 25, 109-110.

Measures to control Toxocara egg contamination in sandpits of public parks.

We investigated measures to prevent Toxocara egg contamination of sandpits. Replacement of contaminated sand was not effective because 1-9 new fecal deposits per sandpit were found daily, with eggs reappearing in the sand 6-9 weeks after the replacement. When the sandpit was covered with a clear vinyl sheet, the temperature of the sand to a depth of 3 cm was 42 degrees C or more for 3 hr when the air temperature was higher than 30 degrees C. This procedure prevented contamination by fecal deposits and also resulted in the destruction of existing eggs because the sandpit was kept dry on rainy days. The method seemed safe and inexpensive. The effectiveness of a fence around a sandpit depended on user behavior, which could not be relied upon. Therefore, a practical method for the prevention of Toxocara egg contamination of sandpits is to cover the sandpit with a clear vinyl sheet at night and on rainy days.

Oligonucleotide synthesis using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-phosphoramidite derivatives.

A comparative study on the utility of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) and 2-(levulinyloxymethyl)benzoyl (LMBz) protecting groups for the 5'-positions of nucleoside 3'-phosphoramidite derivatives in the oligonucleotide synthesis is presented in terms of the syntheses of TpTpT, TpTpTpT, and UpCpApGpUpUpGpG. In addition we describe the synthesis, using the LMNBz protecting group, of the CpCpA terminus triplet of tRNAs bearing exocyclic amino groups with 15N-labeling, and the trimer Gp[A*]pG containing 2'-O-(beta-D-ribofuranosyl)adenosine ([A*]), the latter of which is found at position 64 in the yeast initiator tRNA(Met).

Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed to shear stress.

Morphology and mechanical properties of cultured endothelial cells were measured, using a novel atomic force microscope (AFM) system, developed in our laboratory, in conjunction with an inverted confocal laser scanning microscope. We used this system to examine endothelial cell both in static cultures and exposed to a shear stress of 2 Pa. Initially, the three-dimensional topography of a cell was measured by the AFM and a location was selected for the subsequent measurement of the mechanical response of the cell. The surface of statically cultured cell was smooth. The cell height was not altered by the exposed duration of shear stress. A relationship between external force, F, and the indentation depth, delta, was obtained for several different locations on a cell. This force-indentation response was modelled using a quadratic equation, F = adelta2 + bdelta, indicating that two parameters, a and b, will be constants which are representative of the mechanical response. Endothelial cells cultured at static conditions demonstrated a polygonal shape and less stiff mechanical characteristics around the nucleus compared to those at peripheral regions. The stiffness of the endothelial cells exposed to shear stress increased with the duration time of exposure. At 6-h exposures, the stiffness was higher at upstream side of the cell than the downstream side. However, after 24-h exposure, the stiffness was similar on both sides of the cell. These changes in the stiffness of endothelial cells when exposed to shear stress were suggested to correspond with the distribution of stress fibers in the cell.

Clinical results of transcatheter arterial infusion for uterine cervical cancer.

The effects of transcatheter intraarterial infusion of anticancer drugs on the prognosis of cervical cancer were retrospectively studied. Two or three sessions of transcatheter arterial infusion therapy were performed for 68 patients with primary uterine cervical cancer. The number of patients with stage I, II, III, or IV disease were 13, 22, 24, and 9, respectively. Patients with squamous cell carcinoma comprised 3, 17, 17, and 5 of the respective groups, and the patients with stage I and II disease had either adenocarcinoma or adenosquamous carcinoma, or bulky tumor (>4 cm). The drugs infused were cisplatin (60-70 mg/m2), doxorubicin hydrochloride (30-40 mg/m2), mitomycin (15 mg/m2), and 5-fluorouracil (500 mg/body). They were infused via the bilateral internal iliac arteries. Fifty-eight of the 68 patients (85%) received a radical hysterectomy after transcatheter arterial infusion: 12 of 13 with stage I disease, 21 of 22 with stage II disease, 20 of 24 with stage III disease, and five of nine with stage IV disease. Two patients with stage III disease received radical radiotherapy. The other eight patients (one with stage I disease, one with stage II disease, two with stage III disease, and four with stage IV disease) did not receive an operation after transcatheter arterial infusion because they had distant metastases at the time of operation. Thirty-two of 58 patients (56%) received postoperative radiotherapy. The complete histologic response rates (no active cancer cells) after transcatheter arterial infusion were: 2 of 12 patients with stage I disease, 3 of 21 patients with stage II disease, 5 of 20 patients with stage III disease, and one of five patients with stage IV disease. Tumors with squamous cell carcinoma disappeared at a significantly better rate (10/36, 28%) than did tumors with adenocarcinoma or adenosquamous cell carcinoma (1/22, 5%; p < 0.05). The overall 5-year survival rates of the patients with stages I, II, and III disease were 92.3%, 62.2%, and 71%, respectively. The 5-year survival rates of the patients who underwent surgery with stage I, II, and III disease were 100%, 66.3%, and 71.5%, respectively. Leukocytopenia and thrombocytopenia occurred as an acute complication in 75% and 79% of the patients, respectively. As a late complication, ileus occurred in 7%. Transcatheter arterial infusion may improve the prognosis of patients with cervical cancer without increasing the incidence of late complications.

Chronological SPECT studies of a patient with subacute sclerosing panencephalitis.

We describe here chronological single photon emission computed tomography (SPECT) with N-isopropyl-p-[123I]iodoamphetamine (123I-IMP) of a patient with subacute sclerosing panencephalitis (SSPE) from the early clinical stages. The case, an 8-year-old Japanese girl, was in clinical stage I of the disease on admission. A SPECT study with 99mTc-hexamethylprophyleamine oxime (99mTc-HM PAO) and 123I-IMP revealed hypoperfusion of cerebral blood flow in the bilateral occipital areas and a part of the cerebellum. Initially, no abnormal findings were observed on CT and MRI. Subsequently, however, MRI indicated abnormal signal intensity dominantly in the area of the occipital white matter where hypoperfusion of cerebral blood flow was found by the initial SPECT studies. This abnormal cerebral hypoperfusion appeared to improve after treatment with oral inosiplex and intrathecal interferon-alpha over 6 months of the clinical course. The abnormal signal intensity on the MRI also decreased, but simultaneous cranial CT demonstrated diffuse cortical atrophy and slight ventricular dilatation.

Natural language from function dynamics.

A new approach to syntax and semantics of language is presented as a form of function dynamics, which is studied both analytically and numerically. The iteration of the function dynamics leads to articulation and formation of rules, which depend on each other. A hierarchy of meta-rules as rules of rules also emerges through the iteration when the initial function is suitably embedded. Iteration of a model with dialogue between two function dynamics is shown to generate a higher level structure.

A large myxoma of the right atrium demonstrated by thallium-201.

A rare case of right atrial myxoma in which thallium-201 gave a good delineation of the tumor was presented. In this case, the feeding arteries were seen to be highly developed on coronary arteriogram. The amount of blood containing thallium-201 supplied to the tumor through the feeding arteries was so great that the tumor was considered to be visualized by thallium-201 imaging.

Effect of flow direction on the morphological responses of cultured bovine aortic endothelial cells.

The effect of flow direction on the morphology of cultured bovine aortic endothelial cells is studied. Fully confluent endothelial cells cultured on glass were subjected to a fluid-imposed shear stress of 2 Pa for 20 min and 24 h using a parallel plate flow chamber. Experiments on shear flow exposure were performed for (i) one-way flow, (ii) reciprocating flow with a 30 min interval and (iii) alternating orthogonal flows with a 30 min interval. After flow exposure, the endothelial cells were fixed and F-actin filaments were stained with rhodamine phalloidin. Endothelial cells were observed and photographed by means of a microscope equipped with epifluorescence optics. The shape index (SI) and angle of cell orientation were measured, and F-actin distributions in the cells were statistically studied. Endothelial cells under the one-way flow condition showed marked elongation (SI = 0.39 +/- 0.16, mean +/- S.D.) and aligned with the flow direction. In the case of the reciprocating (SI = 0.63 +/- 0.14) and the alternating orthogonal flows (0.64 +/- 0.14), cells did not elongate so strongly as in the case of one-way flow. Although most cells in the reciprocating flow aligned with the flow direction, the cell axes in the alternate orthogonal flow distributed around a mean value of -45.1 degrees with a large S.D. value. Endothelial cells can be expected to recognise the flow direction, and change their shape and F-actin structure.

Biological Control of Cyclamen Soilborne Diseases by Serratia marcescens Strain B2.

Cyclamen plants were treated with a highly chitinolytic bacterium, Serratia marcescens strain B2, and then challenge inoculated with Rhizoctonia solani sclerotia or Fusarium oxysporum f. sp. cyclaminis conidia. The bacterium suppressed these fungal diseases of cyclamen plants, especially the damping off caused by R. solani, in a greenhouse. Strain B2 survived at approximately 106 to 107 CFU/g in soil for 4 months after the initial application under greenhouse conditions. Chitinolytic enzymes and antifungal low-molecular-weight compounds were present in filtrates of S. marcescens B2, which suppressed germination of R. solani sclerotia in vitro.

Techniques of nasotracheal intubation with the fiberoptic bronchoscope.

When nasotracheal intubation with a fiberoptic bronchoscope is performed, the tube may be blocked in the nasal cavity or larynx, resulting in several complications including epistaxis and hoarseness. We review the causes and complications of tube blockage and discuss optimal techniques for minimizing it.

Seroepidemiology of five major zoonotic parasite infections in inhabitants of Sidoarjo, East Java, Indonesia.

We conducted a seroepidemiological survey of zoonotic parasite infection in inhabitants of East Java, Indonesia. The subjects of the survey were 244 persons selected from visitors to Sidoarjo City Hospital in East Java between May 1992 and October 1993. Ninety-seven had diarrhea and the rest came to the hospital for routine check-ups. All serum samples were tested for antibodies against five zoonotic parasites: Toxoplasma gondii, Entamoeba histolytica, Toxocara canis. Angiostrongylus cantonensis, and Anisakis species. Tests used were enzyme-linked immunosorbent assays (ELISA), latex agglutination (LA) test, indirect fluorescence antibody (IFA) test, hemagglutination (HA) test, and gel diffusion precipitation (GDP) test. Some 64% of the subjects had antibodies to T. gondii. The prevalence of antibodies to E. histolytica varied from 2 to 15% depending on the test, but the true rate was probably the 7% or 8% obtained by the HA and IFA tests. The proportions of subjects with positive results were 63% for T. canis, 17% for A. cantonensis, and 11% for the Anisakis spp. The prevalence of antibodies to T. gondii and T. canis was lower in subjects aged 1 to 9 years than in older subjects, probably because the persons in this group had less time to be infected. Antibody titers to A. cantonensis and the Anisakis spp. were high in the juvenile group, perhaps because recent changes in eating habits have increased opportunities for infection.