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.

Identification and characterization of the human serotonin-4 receptor gene promoter.

The human serotonin-4 (5-HT(4)) receptor gene expression is highly regulated in various tissues. We isolated the human 5-HT(4) receptor gene containing the 5'-flanking region and characterized its promoter. By 5'-RACE (5'-rapid amplification of the cDNA ends) and inverse PCR, multiple transcription initiation sites were identified. The most 5' one (assigned to +1) was 5135 bp upstream to the translation start site. The 500-bp 5'-flanking region contained potential binding sites for transcription factor Sp-1, AP-2, AP-4, and GATA. However, this region lacked TATA- and CAAT-boxes. Transient transfection analyses in human choriocarcinoma T3M-3 (5-HT(4) receptor-positive) and HepG2 (5-HT(4) receptor-negative) cells revealed that the region (-210 to -105) is necessary for the basic and cell-type specific 5-HT(4) receptor gene expression. In addition, untranslated exon 1 contained negative (+112 to +182) as well as positive (+1 to +111) modulators, indicating that exon 1 plays a regulatory role in the 5-HT(4) receptor gene expression.

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.

Functional disorders of the sympathetic nervous system in mice lacking the alpha 1B subunit (Cav 2.2) of N-type calcium channels.

N-type voltage-dependent Ca(2+) channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the alpha(1B) subunit (Ca(v) 2.2). The alpha(1B)-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to omega-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

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.

Preoperative UFT administration for patients with advanced colorectal cancer--increased uptake of 5-fluorouracil by tumor tissue is a prognostic factor.

The aim of this study was to clarify whether increased 5-fluorouracil (5-FU) uptake by tumor tissue following preoperative UFT administration is a prognostic factor after surgery in colorectal cancer patients. We examined the concentrations of 5-FU in tumor or normal tissue of 96 colorectal cancer patients who received UFT (400 mg/day) orally for 7 days prior to surgery. Patients were divided into two groups with high or low 5-FU concentrations in tumor tissue (defined as higher or lower than the cut-off value, respectively). The cut-off value of 5-FU was established based on the upper limit of the 95% confidence interval of the median of the concentration found in normal tissue (0.106 microg/g). Of the 96 patients, 62 (64.6%) were in the low-5-FU group and 34 (35.4%) in the high-5-FU group. The latter had a more favorable clinical outcome (p=0.0465). Cox regression analysis revealed that two independent variables, stage and 5-FU status in tumor tissue, were significant for prediction of survival. These findings suggest that increased uptake of 5-FU by tumor tissue following preoperative oral administration of UFT is an independent prognostic factor in colorectal cancer patients. This variable needs to be considered in the design of future therapeutic trials.

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.

The outer membrane protein I of Pseudomonas aeruginosa PAO1, a possible pollutant of dialysate in hemodialysis, induces cytokines in mouse bone marrow cells.

The finding of outer membrane protein I (OprI) of Pseudomonas aeruginosa in hemodialyzers used by patients with end-stage renal failure led us to study the possible role of OprI as cytokine inducer. However, there are few reports on the biological activity of OprI, because it is difficult to obtain highly purified OprI. In this study, we attempt to establish a procedure for the efficient purification of OprI, which does not include lipopolysaccharide, from the bacterial culture broth, not hemodialyzers, to demonstrate that OprI is a potent cytokine inducer. From bacterial culture broth (1 liter), P. aeruginosa PAO1, which was confirmed previously by the sequence coding, was separated by centrifugation, high-performance liquid chromatography, and disk electrophoresis. Mouse bone marrow cells were stimulated by purified OprI, and the supernatants of the culture were analyzed by several enzyme-linked immunosorbent assay kits. The tumor necrosis factor alpha production stimulated by purified OprI was confirmed and degraded within 24 h. Furthermore, interleukin (IL) 1alpha, IL-1beta, IL-6, and granulocyte-macrophage colony-stimulating factor were also induced by OprI despite the absence of lipopolysaccharide. We conclude that OprI has the potential to induce tumor necrosis factor alpha production in mouse bone marrow cells and that tumor necrosis factor alpha contributes to the induction of inflammatory cytokines, namely IL-1alpha, IL-1beta, IL-6, and granulocyte/macrophage colony-stimulating factor, while lipopolysaccharide has little effect on these cells. These results suggest the presence of a pathway of inflammatory signal transduction triggered by OprI. In addition, OprI is possibly one of the harmful dialysate pollutants in hemodialysis patients besides the well-known lipopolysaccharide.

Further characterization of the 5'-flanking promoter region of the human beta1-adrenergic receptor gene.

Further characterization of the 5'-flanking promoter region of the human beta1-adrenergic receptor (AR) gene was attempted. The transcription initiation sites, determined by the primer extension and the rapid amplification of the 5'-cDNA end, are multiple in a spanning about 30 nucleotides (-289 to -261 relative to the translation start site). There exist inverted CCAAT boxes, multiple binding sites for transcription factor Sp1 and AP-2 nearby transcription initiation sites, however, this region lacks a typical TATA box. In order to localize the regulatory region for the basal transcription of the human beta1-AR gene, a variety of 5'-flanking sequence/chloramphenicol acetyltransferase reporter gene fusion constructs was prepared and transiently expressed in HeLa cells. Functional analyses reveal negatively (-3813 to -2925 and -1772 to -796) as well as positively (-2925 to -1772) regulatory regions, in addition to the region (-796 to -87) being necessary for the basic expression of the human beta1-AR gene.

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.

Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets.

Human and rabbit platelets fully aggregated by platelet-activating factor (PAF) underwent slow disaggregation but were rapidly disaggregated by the PAF receptor antagonists WEB-2086, Y-24180, SM-12502, and CV-3988. Whereas the 1-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine ([3H]acetyl-PAF) specifically bound to platelet receptors underwent slow and spontaneous dissociation, it dissociated promptly from its receptor when WEB-2086 was added, in parallel with platelet disaggregation and disappearance of P-selectin on the cell surface. Extracellular [3H]acetyl-PAF was rapidly deacetylated by normal rabbit platelets; some of the [3H]acetyl-PAF was bound to the cells and a very small amount of [3H]acetate was detected in the cells. In contrast, when 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine was added to the platelets, the radioactivity was rapidly incorporated into the 1-alkyl-2-acyl-sn-glycero-3-phosphocholine fraction. These results indicate that 1) continuous binding of PAF to its receptor is necessary for prolonged platelet aggregation, which may be mediated through an unknown signaling system for a long-term cell response rather than a transient signaling system, and 2) most of the [3H]acetyl-PAF bound to platelets is metabolized extracellularly by ecto-type PAF acetylhydrolase, with the lyso-PAF generated being incorporated rapidly into the cells and converted to 1-alkyl-2-acyl-sn-glycero-3-phosphocholine.

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.

[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.

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.

[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.

[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.

Malignant squamous cell carcinoma arising in a lumbar dermoid cyst. A case report.

A case of malignant transformation to a squamous cell carcinoma in a long-standing lumbar dermoid cyst is described. Progress was slow during 6 years. This type of transformation in a dermoid cyst is extremely uncommon and has never been recorded at this site.

PAF-releasing factor in human serum and inflammatory exudate.

A factor responsible for releasing PAF produced in stimulated human polymorphonuclear leukocytes, which had been previously reported in human serum (Miwa et al. (1992) J. Immunol. 148, 872-880), was also confirmed to be present in inflammatory exudate. PAF-releasing factor, partially purified from human serum, was shown to possess higher affinity for PAF than for triacylglycerol, cholesterol ester, fatty acid or phosphatidylcholine. PAF bound to this factor aggregated washed rabbit platelets to the same extent as that bound to BSA, but was difficult to be hydrolyzed using PAF acetylhydrolase. These observations strongly suggest that PAF-releasing factor functions as a PAF carrier in blood and in the inflammatory response.

Kinetics of epidermal growth factor in saliva.

Human epidermal growth factor (hEGF) stimulates the growth and differentiation of various tissues. We measured EGF levels in saliva (n = 128), urine (n = 94), and serum (n = 99) with radioimmunoassay in order to study the kinetics of hEGF in saliva of normal subjects and patients with oral disease. Salivary EGF levels showed an apparent diurnal rhythm related to the taking of meals. Urinary and serum EGF levels showed no obvious diurnal rhythm. There was no significant correlation between salivary and urinary EGF levels, nor between salivary and serum EGF levels. Salivary EGF levels were significantly lower in the younger group (0-9 years old, 3.06 +/- 0.32 ng/ml, p < 0.05) than in the elder group (10-79 years old, 4.78 +/- 3.5 ng/ml), but did not correlate with age in the elder group. There was no significant difference between males and females between EGF levels in saliva, urine or serum. The relative proportion of EGF levels in submandibular gland saliva, parotid saliva, and whole saliva was 1:6:4. The positive rate of immunohistochemical EGF showed no significant differences between submandibular gland, parotid gland, sublingual gland or minor salivary gland. Salivary EGF levels were markedly low in patients with oral inflammations (stomatitis aphthosa, or peritonsillar abscess) or head and neck tumors (squamous cell carcinoma of the tongue, oral cavity, hypopharynx or larynx). These findings may be significant pathophysiologically. Low salivary EGF levels may reduce the capacity of oral mucosal defense mechanisms to fight against injury by physiochemical agents.