A Single Amino Acid Substitution in the Intervening Region of 129K Protein of Cucumber Green Mottle Mosaic Virus Resulted in Attenuated Symptoms.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

Multiple gene copies for bombyxin, an insulin-related peptide of the silkmoth Bombyx mori: structural signs for gene rearrangement and duplication responsible for generation of multiple molecular forms of bombyxin.

Thirty-eight genes that encode bombyxin, the insulin-related brain secretory peptide of the silkmoth Bombyx mori, have been cloned and characterized. These genes have been classified into four families, A, B, C and D, according to their sequence similarity. All the bombyxin genes lack introns. Five of them have structural features of pseudogenes. The 38 genes cluster in the three DNA segments of Bombyx in unique distribution patterns. Their arrangement has been classified into three categories: gene pairs, gene triplets and single genes. In the pairs, two bombyxin genes belonging to families B and A (B/A) or to families B and C (B/C) are apposed with opposite transcriptional orientation. All triplets are arranged in the order of the family-B, family-C and family-A genes, and the transcriptional directions of the family-C and family-A genes are opposite to the direction of the family-B gene. The bombyxin gene triplets may have been generated by an unequal crossing-over between two gene pairs, B/A and B/C. Crossing-over may have occurred in the bombyxin family-B genes to increase their structural diversity. Duplications may have served to multiply the bombyxin gene triplets. These genomic rearrangements are thought to have led to the generation of multiple bombyxin gene copies and their diversity in structure and genomic organization.

Utility of measuring serum parathyroid hormone-related protein concentration in leukemic patients with hypercalcemia for assessing disease status.

OBJECTIVE: To evaluate serum parathyroid hormone-related protein (PTHrP) as a marker of hypercalcemia in leukemic patients. DESIGN AND METHODS: We measured the serum levels of PTHrP, lactate dehydrogenase (LDH) and calcium in three patients with hypercalcemia due to leukemia. RESULTS: Serum levels of PTHrP, LDH and calcium were elevated at admission in all patients, and these levels were reduced to within the normal range after chemotherapy. However, normalization of serum PTHrP concentration occurred more rapidly than normalization of serum LDH levels after chemotherapy. The increase in serum PTHrP concentration accompanied leukemic cell proliferation and preceded the increases in serum LDH and calcium. Serum LDH concentration increased, but serum PTHrP concentration did not after administration of granulocyte colony-stimulating factor. CONCLUSION: These findings suggest that serum PTHrP may be a more useful marker than serum LDH or calcium in assessing the status of leukemic patients with hypercalcemia.

Differential nociceptive responses in mice lacking the alpha(1B) subunit of N-type Ca(2+) channels.

The role of N-type Ca(2+) channels in nociceptive transmission was examined in genetically engineered mice lacking the alpha(1B) subunit of N-type channels and in their heterozygote and wild-type littermates. In alpha(1B)-deficient mice, N-type channel activities in dorsal root ganglion neurons and spinal synaptoneurosomes were eliminated without compensation by other types of voltage-dependent Ca(2+) channels. The alpha(1B)-deficient mice showed a diminution in the phase 2 nociceptive responses more extensively than in the phase 1 nociceptive responses of the formalin test. The alpha(1B)-deficient mice exhibited significantly increased thermal nociceptive thresholds in the hot plate test, but failed to increase mechanical nociceptive thresholds in the tail pinch test. These results suggest a crucial role of N-type channels in nociceptive transmission, especially for persistent pain like phase 2 of the formalin test and for nociception induced by thermal stimuli.

Tyrosine hydroxylase and monoamine oxidase-A activity increases in differentiating human neuroblastoma after elimination of dividing cells.

SY5Y neuroblastoma cells were treated with retinoic acid to induce differentiation of neuroblast-like cells and with aphidicolin (an inhibitor of DNA polymerase) to eliminate the flat cells present in the long-term cultures and masking some of the biochemical developmental changes. Catecholaminergic enzyme (tyrosine hydroxylase and monoamine oxidase-A) activity was consistently increased with development and the increase was significantly greater after aphidicolin-induced elimination of dividing, non-neuronal cells.

Initial detection of [3H]clonidine binding to solubilized rat brain alpha 2-adrenergic receptors: regulation by guanine nucleotides.

Alpha 2-adrenergic receptors labeled by [3H]clonidine (alpha 2-agonist) can be solubilized from the rat brain with a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). CHAPS-solubilized receptors have the same characteristics of membrane-bound alpha 2-receptors in the brain, and the regulation of receptor binding by guanine nucleotides is retained in the soluble state.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Role of serotonin in the regulation of beta-adrenoceptors by antidepressants.

The role of serotonin (5-HT) in the regulation of the beta-receptor density induced by long-term treatment with typical and atypical antidepressants was examined. Treatment with either mianserin (15 mg/kg, twice daily) or maprotiline (10 mg/kg, twice daily) for 7 days caused a significant decrease in the beta-receptor density, measured 6 h after the last dose, without a change in affinity. The reduction in beta-receptors disappeared rapidly (within 24 h). However, treatment with mianserin or maprotiline combined with fluoxetine, a selective 5-HT uptake inhibitor, significantly decreased the beta-receptor density even 24 h after the last dose. The combined administration of mianserin and 5-hydroxytryptophan (5-HTP) mimicked the effect of the combination with fluoxetine. Following pretreatment with p-chlorophenylalanine (PCPA) for 6 days, desipramine treatment for 3 days significantly decreased the beta-receptors 6 h after the last dose but this desipramine-induced decrease in beta-receptors was rapidly reversible (within 24 h). These results demonstrate that while intrasynaptic 5-HT levels are not a factor in the decrease in beta-receptors, they do play an important role in the preservation of the down-regulated state of the beta-receptor caused by antidepressants from rapid reversibility.

Multicenter randomized prospective study of adjuvant chemotherapy with UFT and mitomycin C in advanced colorectal cancer.

BACKGROUND: A randomized prospective trial was performed to determine the efficacy of preoperative and postoperative adjuvant oral UFT, administered with mitomycin C (MMC) after resection for advanced colorectal cancer. MATERIALS AND METHODS: A total of 126 patients were entered in the study. The patients received UFT (400 mg daily) administered orally for seven days prior to surgery and were randomly assigned to two groups immediately following surgery. Group A received MMC postoperatively; Group B received the same regimen as Group A, plus administration of UFT orally at a dose of 400 mg daily for one year. RESULTS: The survival results revealed no significant difference between groups A and B. In patients with nuclear DNA aneuploid tumors, the hematogenous recurrence rate after curative surgery was lower in Group B than in Group A (P = 0.0656). CONCLUSIONS: Preoperative and postoperative adjuvant oral UFT, administered with MMC after curative resection, may be effective in preventing hematogenous recurrence in colorectal cancer patients with nuclear DNA aneuploidy tumors.

Bombyxin F1 gene: structure and expression of a new bombyxin family gene that forms a pair with bombyxin B10 gene.

Bombyxin F1 gene, a new bombyxin family gene, has been identified. The F1 gene forms a pair with bombyxin B10 gene with an opposite transcriptional orientation and the gene pair F1/B10 is located between bombyxin gene pairs B9/C1 and A7/B7 in a bombyxin gene cluster. The nucleotide sequence of the F1 gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family-A, family-B, family-C, family-D, and family-E bombyxin genes; the bombyxin F1 gene and preprobombyxin F1 share no more than 62% and 53% sequence identities with other bombyxin members, respectively. Harr-plot analysis indicated that the spacer of the F1/B10 gene pair has low sequence similarity with that of other bombyxin gene pairs characterized. The bombyxin F1 mRNA in Bombyx mori brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs. Genomic Southern hybridization indicated that the Bombyx haploid genome contains a single copy of the family-F bombyxin gene.

International Child Care Practices Study: infant sleeping environment.

BACKGROUND: The International Child Care Practices Study (ICCPS) has collected descriptive data from 21 centres in 17 countries. In this report, data are presented on the infant sleeping environment with the main focus being sudden infant death syndrome (SIDS) risk factors (bedsharing and infant using a pillow) and protective factors (infant sharing a room with adult) that are not yet well established in the literature. METHODS: Using a standardised protocol, parents of infants were surveyed at birth by interview and at 3 months of age mainly by postal questionnaire. Centres were grouped according to geographic location. Also indicated was the level of SIDS awareness in the community, i.e. whether any campaigns or messages to "reduce the risks of SIDS" were available at the time of the survey. RESULTS: Birth interview data were available for 5488 individual families and 4656 (85%) returned questionnaires at 3 months. Rates of bedsharing varied considerably (2-88%) and it appeared to be more common in the samples with a lower awareness of SIDS, but not necessarily a high SIDS rate. Countries with higher rates of bedsharing appeared to have a greater proportion of infants bedsharing for a longer duration (>5 h). Rates of room sharing varied (58-100%) with some of the lowest rates noted in centres with a higher awareness of SIDS. Rates of pillow use ranged from 4% to 95%. CONCLUSIONS: It is likely that methods of bedsharing differ cross-culturally, and although further details were sought on different bedsharing practices, it was not possible to build up a composite picture of "typical" bedsharing practices in these different communities. These data highlight interesting patterns in child care in these diverse populations. Although these results should not be used to imply that any particular child care practice either increases or decreases the risk of SIDS, these findings should help to inject caution into the process of developing SIDS prevention campaigns for non-Western cultures.

[Serial changes of MRI and SPECT findings in a case of adult-onset SSPE].

A 20-year-old man who had developed involuntary movement of his left hand and memorial disturbance visited our hospital in December, 1991. On admission, myoclonus, dementia and speech disturbance were recognized. He was diagnosed as subacute sclerosing panencephalitis (SSPE) based on a high titer of serum anti-measles antibody (1/256), serum anti-measles-IgG antibody (> 1/4,800) and typical EEG fiding of periodic synchronus discharge (PSD). Inosine pranobex was administrated orally (4,800mg per day). Serial cranial magnetic resonance imagings (MRI) were taken since January, 1992 to June, 1994. No abnormal finding was demonstrated until April 16, 1992 in MRI, but 123I-IMP SPECT detected decreased accumulation in parietal to occipital lobes on early image in February 5, 1992. Marked high signal area on T2 weighted image in right temporal lobe and parieto-occipital lobe were firstly demonstrated in June 22, 1992 on MRI. These high signal lesions alternated the areas and locations, but the changes were not related to his clinical symptom. These findings may suggest ischemic changes after demyelination. His symptoms have been improving gradually since June, 1994. To our knowledge, 42 cases of adult-onset SSPE were reported so far (5 were in Japan). This case is the first report in the world on adult-onset SSPE serially observed with MRI and SPECT since early stage.

[Paramedian thalamic and midbrain infarcts associated with palilalia].

We report a case with paramedian thalamic and midbrain infarcts associated with palilalia. A 62-year-old man fell into a comatose state, and was admitted to an emergency hospital. Two days later, his consciousness level began to improve. Neurological examination revealed bilateral cerebellar ataxia, oculomotor nerve palsy, lack of spontaneity, and amnesia. Deep tendon reflexes were normal on all extremities, and no muscular weakness was observed. Babinski sign was noted on the right side. Muscle tone was decreased. No sensory disturbance was found. Two months later, he began to show compulsive repetition of syllables, words, or phrases, and was transferred to our hospital. He involuntarily repeated one syllable or a word five to ten times in spontaneous speech, repetition of phrases being not so frequent as that of syllables or words. This speech abnormality was considered as palilalia, which Souques first reported in 1908. The palilalia of this patient was rarely noted when he repeated words spoken to him by the examiner. As he spoke, the rate of speech gradually increased, the loudness reduced, and finally he began to whisper (palilalie aphone). The palilalia continued for four months. MRI showed infarcts in the medial thalami, subthalamic and midbrain on both sides. Auditory brainstem response showed delayed latency of bilateral V waves, and EEG revealed bilateral theta waves. In this patient no lesion other than thalamus, subthalamus and midbrain was detected by MRI. It is suggested that bilateral lesion of the thalami and their projection areas caused palilalia in this patient.

[Intravenous infusion of prostagrandin E1 during and after gastrectomy on postoperative hepato-renal functions in senile patients].

Effect of intravenous prostagrandin E1 (PGE1) during and after surgery on postoperative hepatorenal functions in senile patients was evaluated in 36 elective surgical patients ranged in age from 60 to 85 years. The patients with carcinoma of the stomach underwent total or subtotal gastrectomy under isoflurane anesthesia. These patients were devided into two groups. Eighteen patients received intravenous PGE1 at a rate of 0.03-0.13 micrograms.kg-1.min-1 during surgery and 0.03 microgram.kg.min-1 after surgery until 9:00 am on the first operative day. The remaining 18 patients did not receive PGE1 and served as the control group. Serum GOT and GPT levels in both groups increased significantly at emergence from anesthesia compared with the preanesthetic levels and then declined to the preanesthetic levels on the 3rd postoperative day. Thereafter they increased significantly again on the 7th postoperative day. Serum GOT levels in the PGE1 administered group were significantly lower than those in the control group at the emergence from anesthesia. Serum GPT levels in the PGE1 group tended to be lower than those in the control group on awakening from anesthesia and on the first postoperative day. Serum gamma-GTP levels were stable postoperatively but they increased significantly on the 7th postoperative day in both groups. Serum bilirubin levels were within normal limits in both groups. Postoperative serum levels of urea nitrogen and creatinine were at the preanesthetic levels in both groups. Our findings suggest that continuous intravenous administration of PGE1 during and after surgery is beneficial in attenuating hepatic injury in senile patients for gastrectomy. However, protective effect of PGE1 on postoperative renal function was found to be vague in this study.

[A case of recurrent gallbladder cancer with marked response to arterial infusion chemotherapy and transarterial embolization].

A 50-year-old man with gallbladder cancer was treated by extended cholecystectomy and regional lymph node dissection. At 13 months after surgery, CEA showed high serum levels, and an enlarged liver tumor due to recurrence was demonstrated by computed tomography. After arterial infusion chemotherapy consisting of CDDP, epirubicin and 5-FU, the tumor size and serum level of CEA were significantly decreased. After this therapy and transcatheter arterial embolization, the liver tumor markedly responded and became undetectable. It was suggested that this therapy was effective for gallbladder cancer.

Occurrence of methylguanidine and agmatine in foods.

Methylguanidine (MG), a precursor of carcinogenic methylnitrosocyanamide and methylnitrosourea, has long been considered to occur widely in fresh beef and various fish muscles in fairly high concentrations. Agmatine (AG) has been reported to be easily nitrosated to give a potent mutagen under acidic conditions. A survey has been conducted on the MG and AG contents of various fresh and processed foods. The present study reveals that no appreciable amount, or only trace amounts, of MG could be detected in fresh beef, pork, chicken and various fish and shellfish. In addition, almost the same low levels of MG were detected in various processed foods, except in the case of smoked-dried fish products, where the MG values ranged from 18 to 178 mg/kg. Relatively high concentrations of AG, ranging from 40 to 200 mg/kg, could be detected in fresh abalone and top-shell muscles, while fairly high concentrations of AG could be detected in some processed foods. A dried squid product, in particular, was found to contain as high as 650 mg/kg of AG.