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.

Binding of [35S]saccharin to a protein fraction of rat tongue epithelia.

The binding of [35S]saccharin to ammonium sulfate fractions from homogenates of rat tongue epithelia was measured by equilibrium dialysis. The 40--60% saturated ammonium sulfate fraction from the buffer-soluble fraction had the highest saccharin-binding activity. Binding of [35S]saccharin to the 40--60% ammonium sulfate fraction was inhibited by unlabeled saccharin sodium salt. The inhibition increased with increasing unlabeled saccharin concentration and was nearly complete above 10 mM. [35S]Saccharin binding to the 40--60% ammonium sulfate fraction extracted from the tongue epithelia was inhibited by glucose, lactose and sucrose, while binding to similar fractions from tongue muscle was not affected by these sugars. The inhibition of binding of labeled saccharin to the epithelial fraction increased with increasing glucose concentrations. About 35% of the binding was inhibited by 1 M glucose. No significant difference in the amount of inhibition was seen among the three sugars at 0.1 M. The 40--60% ammonium sulfate fraction from tongue epithelium devoid of taste buds bound much less [35S]saccharin than did a similar fraction from epithelium with taste buds. Binding of [35S]saccharin by the preparation from epithelium devoid of taste buds was not inhibited by glucose. The results provide evidence that the 40--60% ammonium sulfate fraction from tongue epithelia with taste buds contains a protein which binds saccharin and sugars. We hypothesize that it is a sweet taste receptor protein.

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.

In vitro synthesis of Japanese encephalitis virus (JEV) RNA: membrane and nuclear fractions of JEV-infected cells possess high levels of virus-specific RNA polymerase activity.

Japanese encephalitis virus (JEV)-specific RNAs (including 42S RNA) were synthesized in subcellular fractions prepared from infected C6/36 cells. This in vitro RNA synthesis essentially required Mg2+ and four ribonucleotides, and it was enhanced by K+. The amounts of RNA synthesized in vitro (in extracts from JEV-infected cells) increased as a function of time after infection. The RNA-synthetic activity in nuclear fractions was the highest among three kinds of subcellular fractions. Our data showed that nonstructural proteins NS3 and NS5 were membrane-associated proteins. In particular, NS3 was found almost exclusively in the nuclear and membrane fractions. Our results suggest that NS5 and NS3 may play specific role(s) in flavivirus RNA replication.

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.

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.

Isolation of cytokinin binding protein from tobacco leaves by bioaffinity chromatography and its partial characterization.

Cytokinin binding protein was isolated and purified from tobacco leaves by bioaffinity chromatography on a Sepharose column on which benzyladenine (BA), synthetic cytokinin, had been introduced as an affinity ligand by the cyanogen bromide method. The purified protein bound specifically to cytokinins; its binding was inhibited remarkably by the addition of BA and kinetin and slightly by adenine, but not by adenosine in vitro. The dissociation constant, Kd, of the protein-BA complex was about 4 x 10(-5)M. The profiles of SDS polyacrylamide gel electrophoresis and gel filtration indicate that the protein consisted of a single polypeptide chain. Amino acid analysis showed that the protein contained 4 basic, 6 acidic, and 25 neutral amino acids but lacked tryptophan. The molecular weight of the protein was determined to be about 4,000 to 5,000 daltons by gel filtration, SDS polyacrylamide gel electrophoresis and amino acid analysis. The coupling conditions of BA to Sepharose by the cyanogen bromide method are described and discussed.

Smoking-induced body sway and its suppression by periodic saccades.

The effect of cigarette smoking on body sway, the everlasting displacement of the center of gravity while standing upright, was studied and smoking was found to induce an unusual, oscillatory body sway that was higher in frequency and greater in amplitude than that in controls and lasted in general for several minutes after smoking. This characteristic body oscillation was strongly suppressed by periodic, saccadic eye movements. These results suggest that nicotine absorbed into blood during smoking affects the structures in the brain stem related to the regulation of standing posture and the saccadic eye movement.

Effects of periodic saccades on the body sway in human subjects.

The body sway in upright standing was decreased by periodic saccades. The decrease was also observed during voluntary rapid eye movements in complete darkness, indicating that the visual information was not concerned with the decrease of the sway. Continuous eye rotations did not affect the body sway, showing that the repetitive activation of eye muscle proprioceptors was not the cause of the sway reduction. These results suggest that some information related to the execution of saccades may affect the spinal motor system and cause the stabilization of the standing posture.

A silent period in sural muscle occurring prior to the voluntary forward inclination of the body.

A silent period lasting for 300-500 msec was observed in triceps surae muscle when the subject inclined forward from the normal standing. This silent period preceded 100 msec or more the beginning of the body inclination and was terminated by an abrupt refiring in the midst of the body swing. The silence was not preceded by the increase in activity of the sural muscle itself or any other leg muscles studied. H-reflex in soleus was strongly inhibited or disappeared during the silent period, indicating that the excitability of the spinal motoneurons was greatly decreased during this period. These findings suggest that the silent period was not induced by some reflex activity in the spinal cord, but was caused by the inhibitory activity based on a central program on the spinal motoneurons.

Remission of myasthenia gravis: clinical, electrophysiological and immunological studies.

The prognosis of 142 patients with myasthenia gravis (MG) was clinically investigated. Forty-nine (35%) had clinical remission (CR) and 23 (16%) good improvement (GI), while 70 (49%) remained in poor condition. Favorable clinical factors for CR were the onset of MG before the age of 20 years, a pre-thymectomy period of less than one year, and a post-thymectomy period of six years or more. Single-fiber electromyography (SFEMG) showed abnormal jitter in nine (47%) of the 19 CR patients, while abnormal jitter was shown in 13 (81%) of the 16 GI patients. Abnormal jitter in CR patients was correlated with the following clinical factors: complication with the thymoma, a period of three or more years from thymectomy to remission, and a remission period of less than six years. An anti-acetylcholine receptor (anti-AChR) antibody was positive in 12 CR patients (63%) as well as in 13 GI patients (81%). Based upon these facts, we point out that true remission seldom occurs in MG patients, and that there exist clinical features that may favorably induce clinical remission. We would like to postulate that electrophysiological and immunological follow-up is indispensable even in CR patients to predict recurrence.

Biological activities of the structural proteins of Japanese encephalitis virus.

Structural proteins V1, V2 and V3 of Japanese encephalitis virus (JEV) were isolated and purified by means of polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and extracted from the sliced gel. The purified and renatured V3 envelope protein was found to bind to receptors for JEV on red blood cells. V3 also bound to haemagglutination inhibition (HI) antibodies from anti-JEV sera. In addition, V3 protein induced neutralizing antibodies against JEV when injected into mice. These facts strongly suggest that V3 protein carries the antigenic epitope(s) that elicit neutralizing antibodies and react with HI antibodies. We conclude the V3 plays an important role in virus infection. V2 protein did not exhibit these biological activities, but antibody against V1 protein, not contaminated with V3 protein, showed slight virus neutralizing activity. Possible implications of these results are discussed.

Differences in biological activity of the V3 envelope protein of two Japanese encephalitis virus strains.

Two stains Nakayama-NIH and JaGAr-01 of Japanese encephalitis virus (JEV) distinctly differ in their virulence. Amino acid analysis of the V3 viral protein revealed different serine and tyrosine contents in JaGAr-01 V3 as compared to Nakayama -NIH V3. SDS-PAGE purified renatured JaGAr- and Nakayama-V3 proteins could absorb cross-reactive neutralizing antibodies present in anti-nakayama and anti-JaGAr hyperimmune sera. They also absorbed strain specific antibodies of anti-JaGAr and anti-Nakayama sera, respectively. These observations clearly suggest that both JEV strain specific and cross reactive antigenic determinants are present on V3 protein molecules. Antibody against purified Nakayama V3 protein neutralized Nakayama-NIH virus, but had little neutralizing activity against JaGAr-01 virus. The phenomenon is less pronounced when antibodies against Nakayama-NIH virions are used rather than those against purified homologous V3. This suggests that the antigenicity of the purified V3 protein differs from that of the intact virus envelope.

[A form of dopa-responsive dystonia of late onset with diurnal fluctuations].

We report a case of a 67-year-old woman who had dopa-responsive dystonia of late onset with diurnal fluctuations. She was well until the age of 65 years, when she noted the insidious onset of involuntary movements mainly involving the neck and trunk. She had no family history of movement disorders and had never received neuroleptics. Two years after her symptoms began, she visited our clinic. Neurological examination revealed slow repetitive extension and flexion movements of the neck and trunk, and irregular slow movements involving the mouth, tongue and limbs. The cranial nerves, cerebellar function, muscle strength, deep reflexes and sensory function were intact. Clinically and electromyographically, dystonia was characteristic of her involuntary movements. No parkinsonian features were present. The involuntary movements showed diurnal fluctuations that improved after sleep and the administration of L-DOPA and trihexyphenidyl. Dopamine receptor blocking agents aggravated her condition. Routine blood chemistry including copper metabolism, cerebrospinal fluid findings, and brain CT scan were all normal. Dopa-responsive dystonia is characterized by onset in childhood or adolescence and is frequently associated with parkinsonian features. Our patient had non-hereditary neck and trunk dystonia of late onset that responded to L-DOPA. Her disorder may constitute a specific form of dopa-responsive dystonia.

[A case of cranial polyneuropathy presenting with prominent facial diplegia, elevated serum Borrelia burgdorferi antibody and lymphocytic pleocytosis].

A 54 year-old male patient developed acute cranial polyneuropathy including prominent facial diplegia and radicular++-neuritis. He was proven to have lymphocytic pleocytosis, and elevated serum Borrelia burgdorferi antibody to X800 (normal; less than X200). A diagnosis of typical early neuro-borreliosis was made after these clinical and laboratory findings. This case is the first neuro-borreliosis showing the triad of neurological manifestations (meningitis, cranial neuritis, radicular++-neuritis) in Japan. It is concluded that neuro-borreliosis should be considered to be a cause of acute cranial polyneuropathy, particularly of facial diplegia, even if the patient has no apparent history of a tick bite.

[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.

[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.

[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.

[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.