Expression pattern of Galectin 4 in rat placentation.

Galectin 4 (Gal4) is abundantly expressed in the epithelium of the gastrointestinal tract, and functional analysis has concentrated on its roles associated with polarized membrane trafficking. This study aimed to investigate the expression of Gal4 in placentation. The expression level of Gal4 was revealed to be lower in differentiated Rcho-1 cells (a model system of rat trophoblast differentiation) than in proliferative cells. In the rat placenta, immunohistochemical analysis showed that Gal4 is preferentially located in the maternal-fetal junctional zone. These results suggest that down-regulation of Gal4 may be involved in the promotion of trophoblast cell differentiation.

Clonality analysis of lymphoproliferative disorders in patients with Sjögren's syndrome.

The aim of this study was to clarify the nature of the clonal lymphocyte infiltration in Sjögren's syndrome (SS) patients associated with lymphoproliferative disorders. We examined B cell clonality in lymphoproliferative tissues from six primary SS patients associated with lymphoproliferative disorders or lymphoma by cloning and sequencing of the gene rearrangement of the immunoglobulin heavy chain complementarity determining region 3 (IgVH-CDR3). Three patients with sequential observation showed progressional clonal expansion with the presence of the same subclone in different tissues during the course of disease. Among them, one patient developed mucosa-associated lymphoid tissue (MALT) lymphoma in glandular parotid. The other three SS patients concomitant with malignant B cells lymphomas showed different clonal expansion of B cells between nodal sites and salivary glands. The cloanality analysis indicated that monoclonal B cell population could spread from one glandular site to another site during the course of SS, suggesting that the malignant clone may arise from the general abnormal microenvironment, not restricted to the glandular tissue, in some SS patients.

Cyclic changes of granulocyte colony-stimulating factor (G-CSF) mRNA in the human follicle during the normal menstrual cycle and immunolocalization of G-CSF protein.

BACKGROUND: Ovulation has several similarities with inflammation and is closely connected to the activity of leukocytes and inflammatory cytokines. Since granulocytes are one of the major leukocytes, we focused our attention on the presence and local production of granulocyte colony-stimulating factor (G-CSF) in the human ovary. METHODS: The presence of G-CSF protein in the follicular fluid and perifollicular tissues was examined by Western blot analysis (n = 5) and immunohistochemical staining (n = 10). The relative expression levels of G-CSF mRNA in relation to GAPDH in granulosa, theca and luteal cells during the menstrual cycle were measured by quantitative RT-PCR using TaqMan technology (n = 15). RESULTS: G-CSF protein was detected in all follicular fluid and located mainly in granulosa cells of the follicle and luteal cells. The expression level of G-CSF mRNA in the late follicular phase was 137.6 +/- 18.5, which was approximately 10-fold greater than other phases during the menstrual cycle (P < 0.05). CONCLUSIONS: These results demonstrate that G-CSF is produced in the human follicle shortly before the ovulatory phase and may play an important role in the mechanism of ovulation.

Expression of multidrug resistance protein and messenger RNA correlate with (99m)Tc-MIBI imaging in patients with lung cancer.

UNLABELLED: In vitro studies have shown that (99m)Tc-sestamibi (MIBI) is a transport substrate for the P-glycoprotein (Pgp) pump and the multidrug resistance-associated protein (MRP) pump. However, whether MRP and lung resistance protein (LRP) affect tumor accumulation and efflux of (99m)Tc-MIBI in lung cancer is not known. In this study, we explored whether Pgp and the other pumps, MRP and LRP, affect tumor accumulation and efflux of (99m)Tc-MIBI in lung cancer. METHODS: Thirty-four lung cancer patients who underwent surgery were examined. Before surgery, (99m)Tc-MIBI SPECT was performed 15 min and 180 min after injection, and early uptake, delayed uptake (L/Nd), and washout rate (L/Nwr) of (99m)Tc-MIBI were obtained. Pgp, MRP, and LRP expression were investigated by immunohistochemistry. The messenger RNA (mRNA) level of Pgp, MRP, and LRP was determined by real-time reverse-transcription polymerase chain reaction. The lung cancer (99m)Tc-MIBI images were correlated with protein and mRNA expression. RESULTS: The mean L/Nd of the Pgp (-) group was significantly higher than that of the Pgp (++) group (P = 0.0324). The Pgp (++) group had a higher L/Nwr than did the Pgp (-) group (P = 0.0269). The mean L/Nd of the Pgp mRNA low-expression group was significantly higher than that of the Pgp mRNA high-expression group (P = 0.0127). The Pgp mRNA high-expression group had a higher L/Nwr than did the Pgp mRNA low-expression group (P = 0.0825). No appreciable correlation was found between the lung cancer (99m)Tc-MIBI images and the expression of MRP or LRP on the level of protein or mRNA. CONCLUSION: These data suggest that an increased level of Pgp expression correlates with a low accumulation on delayed scans and a high L/Nwr of (99m)Tc-MIBI in lung cancer. Neither MRP nor LRP expression on the level of either protein or mRNA correlated significantly with tumor accumulation or efflux of (99m)Tc-MIBI in lung cancer.

Comparative sequences of two type 1 dengue virus strains possessing different growth characteristics in vitro.

The complete genome sequences of two dengue-1 virus strains having different growth characteristics (Mochizuki and A88) were compared with other published strains. The sequence analysis indicated several unique amino acid changes throughout the coding region of Mochizuki strain, mostly in envelope (E) protein. A unique amino acid, Ile-69 for Mochizuki strain at E protein resulted in the loss of an Asn-67-linked glycosylation site. A Thr substitution for Ala-114 at C protein and amino acid changes found in E, non-structural NS3, NS4a, and NS5 proteins were unique for A88 strain. These substitutions might be correlated to their different growth characteristics in vitro.

[Extraction of RNA from paraffin embedded tissues and analysis of p53 gene expression in colonic cancer].

We examined, immunohistochemically and molecular biologically, p53 gene expression in 10 patients with colonic cancer. RNA was extracted from paraffin embedded normal and colonic cancer tissues by using RNA isolator kit and proteinase K. The most effective time and concentration of proteinase K for RNA extraction was 24 hours and 100 micrograms/ml, respectively. P53 gene expression was analyzed by ABI PRISM 7700 Sequence Detection System(ABI 7700 System, Perkinelmer). Gene expression level in each sample was estimated on the basis of the standard curve of ABI 7700 System. Human G3PDH gene was used as the internal control. Immunohistochemically, the tumor cells in all examined cases showed a strong positivity for anti-p53 gene antibody. In ABI 7700 System, expression of p53 gene in the malignant tissues revealed a high level in only 2 cases that had a clinical stage IV, however, in remaining 8 cases a clinical stage was I to III and expression level of p53 was relatively lower. These results suggest that colonic cancer cells show mutant-p53 gene expression, and a ratio of mutant- to wild-p53 gene may have something to do with a relationship between gene expression and clinical stage.

Lactate discrimination incorporated into echo-planar spectroscopic imaging.

A technique for discriminating a lactate signal from overlapping lipid signals in (1)H spectroscopic imaging is presented. It is based on J-coupling between lactate protons and on the broad spectral bandwidth of lipid signal. Measurement parameters used in the technique are determined so that TE is separated from n/J (n: a natural number, J: J-coupling constant) enough to suppress the lipid signal at the time when the lactate signal is strongest. Data processing is used to calculate the lactate signal intensity from the reconstructed spectra. This technique enables lactate to be discriminated in a single measurement and enables spectra of other metabolites to be acquired simultaneously. However, it necessitates a homogeneous magnetic field, long TE, and supplementary lipid suppression. Discrimination of the lactate signal is demonstrated by applying lactate-discriminating echo-planar spectroscopic imaging (EPSI), which combines this discrimination technique with the standard EPSI, to rat focal cerebral ischemia models. Magn Reson Med 45:568-574, 2001.

Mismatch between lactate and the apparent diffusion coefficient of water in progressive focal ischemia.

In this study, we examined mismatch in the area indicated by the normal apparent diffusion coefficient (ADC) of water and increased lactate in the early stage of focal cerebral ischemia. Five rats were subjected to permanent middle cerebral artery (MCA) occlusion. Diffusion-weighted echo planar imaging (DWEPI) and proton echo planar spectroscopic imaging (EPSI) were performed from 20 to 170 min after MCA occlusion, and lactate and N-acetyl asparate images were obtained by EPSI. Postmortem histological analysis was also performed. The areas of increased lactate and normal ADC were observed in the surrounding border zone of ischemia at approximately 20 min after MCA occlusion. This initial lactate in the border zone was significantly higher than that in the normal area, but lower than that in the ischemic core, which showed a reduction of ADC. However, this area was progressively involved in the ischemic core at 170 min without any treatment. The lactate-ADC mismatch in the initial period of ischemia may offer unique diagnostic information for ischemic tissue at high risk, followed by progressive involvement in the ischemic core without treatment. Considering that the accumulation of initial lactate in this area was not excessive, our findings may suggest that the lactate-ADC mismatch in the early period of ischemia indicates potentially salvageable tissue at high risk, requiring aggressive treatment.

An axonal form of Charcot-Marie-Tooth disease showing distinctive features in association with mutations in the peripheral myelin protein zero gene (Thr124Met or Asp75Val).

OBJECTIVES AND METHODS: Seven families were studied with an axonal form of Charcot-Marie-Tooth disease (CMT) associated with mutations in the peripheral myelin protein zero (MPZ) gene-Thr124Met or Asp75Val. RESULTS: Patients with these mutations commonly showed relatively late onset sensorimotor neuropathy predominantly involving the lower limbs. Sensory impairment typically was marked, and distal muscle atrophy and weakness were also present in the legs. Adie's pupil and deafness were often present, and serum creatine kinase concentrations were often raised irrespective of which MPZ mutation was present. Relatively well preserved motor and sensory nerve conduction velocities contrasted with reduced or absent compound muscle action potentials and sensory nerve action potentials. Axonal change with marked axonal sprouting was seen in sural nerve specimens. CONCLUSION: The similar associated clinical findings suggest that patients with axonal CMT with an MPZ gene mutation share distinctive clinical features.

[Two families of Charcot-Marie-Tooth disease with Adie's pupil, axonal neuropahy and the Thr124Met mutation in the peripheral myelin protein zero gene].

We reported two families of Charcot-Marie-Tooth disease (CMT) with Thr124Met mutation in the peripheral myelin protein zero (MPZ). The clinical features of the proband patients of both families showed Adie's pupil, severe sensory dominant neuropathy in lower extremities, and axonal changes in sural nerve biopsies and nerve conduction studies. Muscle atrophy and weakness was mild in the lower legs, while sensory impairment was marked. The proband patient of family 1 had four symptomatic siblings and one of them showed Adie's pupil. The elderly daughter of the proband of family 2 showed Adie's pupil and younger daughter showed photophobia. The biopsied sural nerves of both proband patients revealed prominent axonal sprouting, and sub-perineurial edema and mild fascicular enlargement. Segmental demyelination was not frequent in teased fiber assessment. The present two family cases strongly suggest that this MPZ gene mutation (Thr124Met) could be present among the patients with CMT type 2, axonal form. Furthermore, the patients showing sensory neuropathy and Adie's pupil may need to be reexamined with this mutation. It is also necessary to reassess genotype-phenotype correlation in CMT patients particularly in reference to type 1 and type 2.

Molecular characterization of the Japanese encephalitis virus representative immunotype strain JaGAr 01.

We determined the full genomic sequence of the Japanese encephalitis virus JaGAr 01 strain and its predicted amino acid sequence. Nucleotide sequence comparison with ten fully sequenced JE strains shows a homology range from 89.62 to 99.49%. Amino acid sequence homologies range from 96.85 to 99.74%. Comparison of amino acid sequences shows a unique amino acid, arginine, for JaGAr 01 at position 123 of the E-protein, while the eight other strains contained serine. Secondary structure prediction by free energy minimization shows a unique structure for JaGAr 01 that includes an RNA segment that is conserved for all flaviviruses. Speculation is made about the role these results may play in the replication and antigenic characteristics of JaGAr 01. Phylogenetic analyses of the E-protein of JaGAr 01 together with 35 other JE strains showed diversity in amino acid characteristics between the prototype strains Nakayama, JaGAr 01 and Beijing-1. Phylogenetic trees computed by neighbor joining and Fitch Margoliash analysis of nucleic acid and protein sequences showed Nakayama and Beijing in one cluster different from JaGAr 01.

[Study of the PCR conditions on a slide for in situ PCR].

In order to find the conditions for in situ PCR, we examined the efficiency of PCR in a solution phase on the slide. DNA was extracted from the cells transfected with HCV-NS3 region. The primers were HCV-NS3 specific oligonucleotides. The results showed that the condition of DNA denaturation on the slide was suited at 90-92 degrees C for 10-60 seconds. In case of over 94 degrees C, DNA was not amplified and it suggests that the activity of Taq-polymerase was lost during the denaturation. As regards annealing temperature and times, if the melting temperature (Tm) was used for the annealing, we needed relatively longer times, and if annealing temperature was about 5 degrees C lower than the Tm, it was enough to use relatively short times. In addition, a favorable result was obtained when in situ PCR was carried out under the best conditions for PCR on a slide. It is indicated that the conditions for general PCR in a tube is not applicable to the amplifying of cellular DNA targets on a slide for detection in situ.

Inhibitory effect of furanonaphthoquinone derivatives on the replication of Japanese encephalitis virus.

Japanese encephalitis still occurs in endemic and epidemic forms over a wide area of Asia. Although the vaccine against Japanese encephalitis virus (JEV) is widely used, no antiviral drug has been reported. We used several different kinds of furanonaphthoquinone derivatives and found antiviral activity against JEV. Especially, 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) indicated the highest antiviral activity, followed by 2-(1-hydroxyethyl)-, 5(or 8)-hydroxy-, and 2-methyl-5(or 8)-hydroxy-analogs of naphtho[2,3-b]furan-4,9-dione. In the presence of 3 microg/ml FNQ3, the virus yields in Vero cells were 2 x 10(5) PFU/ml at 24 h after infecting with the virus and 10% of the control level. Western blot analysis using anti-E rabbit sera or anti-NS3 showed that the expression of viral proteins was inhibited by treatment with FNQ3. In addition, Northern blot analysis indicated that the appearance of JEV-RNA was also inhibited by FNQ3. These results suggest that FNQ3 inhibits JEV replication through viral RNA and protein synthesis.

Effect of bafilomycin A1 on the growth of Japanese encephalitis virus in Vero cells.

We studied the effect of bafilomycin A1 (Baf-A1), a novel and highly specific inhibitor for vacuolar-type proton (V-H+) pump, on the growth of Japanese Encephalitis virus (JEV) in Vero cells. Viral fluorescence microscopic study showed that Baf-A1 induced the complete disappearance of acidified compartments such as endosomes and lysosomes in Vero cells by the treatment with 0.1 microM Baf-A1 for 1 h at 37 degrees C. In proportion to the disappearance of acidified compartments, virus growth was inhibited when Baf-A1 was present from 1 h before infection to the end of incubation in a dose-dependent manner, or added within as early as 5 min after infection. Conversely, the virus growth was recovered in correlation with the reappearance of acidified compartments after removal of Baf-A1. These results suggest that a low pH condition, which is regulated by Baf-A1-sensitive V-H+ pumps, is essential for the early stage of JEV growth.

[Pathologic diagnosis on bone and soft tissue tumors by molecular biological methods].

Characteristic chromosome aberrations and the rearranged genes resulting in chimeric fusion genes have been reported in some bone and soft tissue tumors; t(X; 18) in synovial sarcoma, t(11; 22) in Ewing's sarcoma and primitive neuroectodermal tumor, and t(2; 13) in alveolar rhabdomyosarcoma. We practically used the chromosome analysis and the reverse transcription-polymerase chain reaction (PCR) method as a tool for diagnosis and follow up. All of 10 cases of synovial sarcoma had a chimeric product of SYT/SSX gene. Eleven cases of Ewing's sarcoma and primitive neuroectodermal tumor showed 6 variants of chimeric products between EWS gene and Fli1 gene in the PCR-directed sequence analysis. Although PAX3/FKHD or PAX7/FKHD transcripts were amplified in alveolar rhabdomyosarcoma cases, MyoD1 and myogenin gene which are myogenic transcription factor were also expressed in most rhabdomyosarcomas. These findings indicate that molecular biological analysis may be a useful supplementary method for pathologic diagnosis of bone and soft tissue tumors.