Relative fitness of females and hermaphrodites in a natural gynodioecious population of wild radish, Raphanus sativus L. (Brassicaceae): comparison based on molecular genotyping.

In many gynodioecious species, sex determination involves both cytoplasmic male-sterility (CMS) genes and nuclear genes that restore male function. Differences in fitness among genotypes affect the dynamics of those genes, and thus that of gynodioecy. We used a molecular marker to discriminate between hermaphrodites with and without a CMS gene in gynodioecious Raphanus sativus. We compared fitness through female function among the three genotypes: females, hermaphrodites with the CMS gene and those without it. Although there was no significant difference among the genotypes in seed size, hermaphrodites without the CMS gene produced significantly more seeds, and seeds with a higher germination rate than the other genotypes, suggesting no fitness advantage for females and no benefit to bearing the CMS gene. Despite the lack of fitness advantage for females in the parameter values we estimated, a theoretical model of gynodioecy shows it can be maintained if restorer genes impose a cost paid in pollen production. In addition, we found that females invest more resources into female reproduction than hermaphrodites when they become larger. If environmental conditions enable females to grow larger this would facilitate the dynamics of CMS genes.

Development and characterization of microsatellite markers in Zanthoxylum ailanthoides (Rutaceae).

Nine microsatellite markers were developed for Zanthoxylum ailanthoides, a typical pioneer tree. Averaged over the nine loci, the number of alleles per locus was 5.1. The observed and expected heterozygosities ranged from 0.233 to 0.833 and from 0.314 to 0.823, with averages of 0.606 and 0.641, respectively. No loci showed significant departures from Hardy-Weinberg equilibrium or linkage equilibrium after Bonferroni correction (P > 0.05). These markers will be useful for parentage analyses and studies of population genetic structure in the species.

High population differentiation and unusual haplotype structure in a shade-intolerant pioneer tree species, Zanthoxylum ailanthoides (Rutaceae) revealed by analysis of DNA polymorphism at four nuclear loci.

Differences in demographic history, life-history traits, and breeding systems affect nucleotide variation patterns. It is expected that shade-intolerant pioneer tree species have different patterns of genetic polymorphism and population structure than climax species. We studied patterns of nucleotide polymorphism at four putative starch pathway loci (agpSA, agpSB, agpL, and GBSSI) in Zanthoxylum ailanthoides, a shade-intolerant pioneer tree species that occupies forest gaps in warm-temperate forests of East Asia. Genetic diversity was lower within each population than among populations, and differentiation among populations was high across the loci (F(ST) = 0.32-0.64), as expected from the insect-pollinated breeding system and the metapopulation structure of this pioneer species. Numbers of haplotypes were smaller than those expected from the observed numbers of segregating sites. Single haplotypes accounted for more than 47% of all the sampled genes at the respective loci. These variation patterns were incompatible with neutral predictions for populations of a finite island model. Complex population dynamics, such as bottleneck and/or admixture, in the history of this pioneer tree species might have resulted in the observed patterns of genetic variation and population structure, which are different from those of climax wind-pollinated tree species, such as conifers. In contrast to the other loci investigated in this study, agpL showed nearly no variation in Z. ailanthoides (one singleton only), but there was some extent of variation in a closely related species, Zanthoxylum schinifolium. This suggests possibly a recent selective sweep at or near the locus in Z. ailanthoides.

Variation of female frequency and cytoplasmic male-sterility gene frequency among natural gynodioecious populations of wild radish (Raphanus sativus L.).

In gynodioecious plant populations, sex determination often involves both cytoplasmic male-sterility (CMS) genes and specific nuclear genes that restore male function. How gynodioecy is maintained under the joint dynamics of CMS and restorer genes remains controversial. Although many theoretical models deal with interactions between CMS genes and restorer genes with sexual phenotypes and predict changes in their frequencies, it is difficult to observe the frequencies because no molecular markers have been established for either CMS or restorer genes in well-studied gynodioecious plants. This is the first report of the frequency of a CMS gene determined using a molecular marker in natural populations of a gynodioecious plant. Using a set of CMS gene-specific polymerase chain reaction primers, we compared female and CMS gene frequencies in 18 natural populations of Raphanus sativus. Female frequency was relatively low, ranging from 0 to 0.21. In contrast, the CMS gene frequency was highly variable among populations, ranging from 0 to 1. Estimated restorer gene frequency seemed less variable than observed CMS gene frequency, probably due to higher gene flow than in the CMS gene. Genetic drift may play a role in maintaining high variability of the CMS gene, although other possibilities are not excluded.

Effects of virus infection and light environment on population dynamics of Eupatorium makinoi (Asteraceae).

We studied the effects of virus infection on dynamics of three Eupatorium makinoi populations in contrasting light environments, Gora-dani (a shaded population) and Minou 1 and Minou 2 (open-site populations). Censuses of the plants were taken for 8 yr in Gora-dani and 4 yr in Minou 1 and Minou 2. After the epidemics of virus infection, most plants were virus infected at both sites. The number of plants and the proportion of flowering individuals decreased rapidly and simultaneously in the shaded population in Gora-dani. By contrast, in the open-site populations of Minou, the proportion of flowering plants decreased first, and then the number of plants decreased gradually. Growth analysis of the plants in the Gora-dani population revealed that stem growth was significantly suppressed by infection and that flowering and survivorship of the infected plants decreased with reducing plant height. Since light availability affected plant growth and thereby flowering and survivorship, the differences in population dynamics between the two field sites could be caused by the differences in light environments. Although populations in open sites may persist for considerable periods after virus epidemics, the individual local populations of E. makinoi would eventually become extinct irrespective of light environments.

Maternal control of sex ratio in Rana rugosa: evidence from DNA sexing.

Parental control of primary sex ratio has been reported in a mammal (red deer), some birds, and a snake. However, it remains uncertain whether other vertebrates including Amphibia can control sex ratio. In this paper, we examined the possibility in a wild population of the Japanese frog Rana rugosa which has female heterogamety. Sex ratios of their eggs were determined using DNA markers. The eggs were sampled in the field from May to August in 1998. Each egg was then sexed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using a sex-specific DNA marker. The result showed a male bias early in the season which changed to a female bias later, suggesting that females of R. rugosa can control the primary sex ratio.

Administration of opiate receptor antagonist inhibits mucosal atrophy of the gut in fasting rats.

OBJECTIVE: The objective of this study was to determine whether the opiate mu receptor antagonist naloxone would prevent atrophy of the gut in 24-h-fasted rats. METHODS: Male Sprague-Dawley rats (n = 76, body weight 200-225 g) were catheterized in the jugular vein on Day 0. The rats were fed standard rat chow for 4 days. On Day 4, the diet was changed to the standard liquid diet, and the rats were allowed free access to the liquid diet. On Day 7, the rats were randomized into five groups: (1) free fed, (2) free fed plus naloxone, (3) pair fed, (4) fasting, (5) free fed plus morphine, (6) fasting plus naloxone. Either naloxone (0.16 mg/kg/h) or morphine (0. 21 mg/kg/h) was continuously infused via venous catheter for 24 h. On Day 8, 24 h after fasting or free feeding, the animals were sacrificed. RESULTS: Twenty-four hours of fasting caused atrophy of the jejunum and elevated morphine levels in the brain (free fed, 931. 3 +/- 122.3 fmol/g, vs fasting, 1419.0 +/- 150.0, P < 0.05). Morphine infusion reduced villus height, mucosal weight, and protein content in jejunum as compared with the free fed rats receiving saline. Administration of naloxone caused an increase in villus height (fasting, 587.0 +/- 25.8 microm, vs fasting plus naloxone, 670.0 +/- 17.4, P < 0.05), mucosal weight (fasting, 17.4 +/- 1.8 mg/cm, vs fasting plus naloxone, 22.6 +/- 1.9, P < 0.05), and protein content (fasting, 13.5 +/- 0.7 microg/cm, vs fasting plus naloxone, 16.7 +/- 0.6, P < 0.05) in jejunum. CONCLUSION: Mucosal atrophy of the jejunum is caused by endogenous opioid in fasting rats.

Effect of surgical stress on endogenous morphine and cytokine levels in the plasma after laparoscopoic or open cholecystectomy.

BACKGROUND: Endogenous morphine in the brain leads to various biological responses after surgery. The aim of this study was to determine whether morphine levels in the plasma would be enhanced by open laparotomy rather than by laparoscopic procedures. METHODS: We compared 19 patients who underwent laparoscopic cholecystectomy with five patients who underwent resection of the gallbladder by open laparotomy. Morphine levels in the plasma were measured by an electrochemical detection system. RESULTS: Postoperative endogenous morphine levels were higher with open laparotomy than with the laparoscopic technique (three h after surgery: open, 200 +/- 52.6 fmol/ml vs laparoscopy, 17.6 +/- 3.7, p < 0.01). This morphine elevation accounted for higher levels of cytokine, greater pain scores, and longer duration of fasting in open laparotomized patients than in laparoscopic cholecystectomy patients. Stress hormone levels in the plasma were also higher with open laparotomy than with laparoscopy. CONCLUSION: Morphine synthesis was enhanced by open laparotomy, resulting in greater biological response postoperatively than that seen with laparoscopic cholecystectomy.

Trade-offs between flower number and investment to a flower in selfing and outcrossing varieties of Impatiens hypophylla (Balsaminaceae).

Floral resource allocation was compared on a whole-plant basis between two varieties of Impatiens hypophylla that differ in flower size. There were significant negative correlations between flower number and investments to a flower at both the within-population and between-variety levels. In individual flowers, var. hypophylla with larger flowers invested significantly more resources to male and pollinator-attractive functions, whereas investments to female function did not differ between the varieties. In experimental populations placed in the field, pollinators preferred the larger flowers of var. hypophylla even within the same habitat of var. microhypophylla, which has smaller flowers. There was a significant lack of observed heterozygosity only in var. microhypophylla. Thus, the outcrossing variety had more attractive but fewer flowers, while the selfing variety had less attractive but more abundant flowers.

[Examination of soluble cell adhesion molecules in pregnancy induced hypertension].

This study was performed to determine whether or not the soluble-intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1) and endothelial leukocyte adhesion molecule-1 (sELAM-1) are sensitive markers of pregnancy induced hypertension (PIH). sICAM-1 concentrations were significantly higher (p < 0.05) in the mild PIH compared to non-pregnant women and normal pregnant groups. sVCAM-1 concentrations in the mild PIH group and the severe PIH group were significantly higher than the non-pregnant women group (p < 0.0001, p < 0.01, respectively) and the normal pregnant group (p < 0.0001, p < 0.0005, respectively). The concentrations of sELAM-1 in the mild PIH group were also significantly higher compared to normal pregnant group (p < 0.01). Our results suggest that soluble cell adhesion molecules may be useful markers detecting endothelial damage and dysfunction in patients with PIH.

Effect of intracerebroventricular injection of tumor necrosis factor alpha on gut mucosal protein turnover in mice fed enterally.

OBJECTIVE: The objective of this study was to determine whether mucosal protein turnover is enhanced by intracerebroventricular (ICV) injection of tumor necrosis factor alpha (TNF-alpha) in mice fed enterally. METHODS: Male B6C3F1 mice (n = 37, 20.0-25.0 g) were catheterized in the ICV space using stereotaxic coordination, and subsequently a catheter was inserted into the stomach for enteral feeding on Day 0. The animals were fed a standard mouse diet and water ad libitum for 4 days. On Day 4, enteral feeding was begun with a standard liquid diet, and either artificial cerebrospinal fluid (control) or recombinant human (Rh) TNF-alpha (5 ng/h) was continuously given via the ICV catheter using an osmotic pump. On either Day 5 or 7, the mice were sacrificed 10 min after injection of [U-(14)C]phenylalanine (5 microCi/mouse, ip). Fractional synthetic rates (FSRs) of jejunal mucosa and muscle were measured by the pool flooding technique. Interleukin-6 (IL-6) levels in the plasma were measured by ELISA. RESULTS: Body weight was significantly reduced by ICV injection of TNF-alpha, because muscle FSR was decreased with TNF-alpha. There were no differences in IL-6 levels in the plasma between the two groups. The FSR of jejunal mucosa was elevated by infusion of TNF-alpha as compared with control mice on both Days 5 and 7 [Day 5: (control) 99.5 +/- 17. 6%/day vs (TNF) 150.5 +/- 20.5%/day, P < 0.05; Day 7: (control) 54.8 +/- 8.8%/day vs (TNF) 102.7 +/- 19.1%/day, P < 0.05). CONCLUSION: injection of TNF-alpha enhanced the mucosal protein synthesis rate without elevating IL-6 levels as compared with control mice, suggesting that the central nervous system links to the gut to regulate mucosal protein turnover through cytokine in the brain, but plasma IL-6 is not involved in this linkage.

Molecular evolution of a host-range gene in geminiviruses infecting asexual populations of Eupatorium makinoi.

Asexual plants of Eupatorium makinoi is frequently infected with tobacco leaf curl geminivirus (TLCV). The host range of TLCV is narrow, and ORF C4 is considered to function as a host range determinant. Using this TLCV-Eupatorium system, we tested the expectation that the rate of amino acid replacements will be accelerated in ORF C4 if resistant genes of the host plants drive molecular evolution in ORF C4. ORF C4 is entirely contained within a longer ORF C1 encoding a replication protein. We analyzed 21 sequences containing ORF C4 and a part of ORF C1. While per-site number of synonymous substitutions exceeded that of replacements in ORF C1, per-site number of replacements exceeded that of synonymous substitutions in ORF C4. However, this excess of per-site replacement in ORF C4 was mostly explained by the overlap gene nature, because most synonymous substitutions in ORF C1 change amino acid of ORF C4. In conclusion, not positive but negative selection is a predominant mode characterizing molecular evolution of ORF C4.

Effects of variation in flower number on pollinator visits in Cirsium purpuratum (Asteraceae).

We examined the functional relationships between floral display and two types of bumble bee response, the visitation rate per plant and the number of flowers visited on a plant, in an artificially arranged field population of Cirsium purpuratum. To reduce the variance in data, we collected data for each day separately and adopted a Latin square design in selecting the focal plants within a day. We then tested several types of regressions to each set of data to find the best-fitting line accounting for the observed relationship between pollinator response and display size. We found that the visitation rate of bumble bees per plant was a decelerating function of floral display, and that the number of flowering heads visited on a plant increased linearly with display size. Predicted from the above two functions, the visitation rate per head was independent of floral display and nearly constant within each day. Our results suggest that conventional methods in collecting and analyzing data on pollinator visitation may yield large variance in data derived from temporal and spatial heterogeneity and that improved methods employed here are effective in reducing the variance and estimating patterns of pollinator response to floral display more accurately.

[A case of congenital complete heart block in a mother with anti-52 kD SS-A/Ro antibodies].

We report a case of congenital complete heart block (CCHB). A 38-year-old woman was admitted our hospital because of fetal bradycardia at 21 weeks 3 days of gestational age. She had no symptom of collagen disease. On admission, laboratory data showed positive anti-nuclear antibodies, anti-SS-A/Ro antibodies and anti-52 kD SS-A/Ro antibodies. But anti-60 kD SS-A/Ro and anti-SS-B/La antibodies were negative. Consequently anti-52 kD SS-A/Ro antibodies positive woman had an infant with CCHB. The baby was equipped with pacemaker at the age of 2 months. This report suggests that anti-52 kD SS-A/Ro antibodies may play an important role in the development of CCHB.

Effects of virus infection and growth irradiance on fitness components and photosynthetic properties of Eupatorium makinoi (Compositae).

We examined the effects of geminivirus infection on fitness components and on photosynthetic properties of the host plant, Eupatorium makinoi, grown at two irradiance levels in a natural-light greenhouse. Under the low-light condition (13% full sunlight), more than a half of the infected plants died during the 9-mo experiment, while most of uninfected plants survived. Growth rate was also lowered by infection. At high light (50% full sunlight), by contrast, virus infection did not cause mortality despite slight decrease in growth rate. Flowering occurred only at high light, and reproductive outputs of the plants were markedly reduced by the infection. Infected leaves had distinct yellow variegations and, when compared with uninfected leaves, they showed (1) comparable light-saturated photosynthetic rate per unit area, but (2) lower initial slope of light-response curve of photosynthesis on an incident irradiance basis. The lower initial slope was mainly due to reduction of light-harvesting chlorophyll-protein complexes in the variegated parts. Since the differences in plant performance, depending both on infection and on growth irradiance, were largely explained by the differences in growth rate and/or plant size, the reduced photosynthetic production in the infected plants would be a major factor explaining the inferior performance of the host plants.

Thymidylate synthase inhibition by an oral regimen consisting of tegafur-uracil (UFT) and low-dose leucovorin for patients with gastric cancer.

PURPOSE: 5-Fluorouracil (5-FU) remains a standard therapy for patients with advanced gastric cancer. There has been no study using an oral regimen with a combination of tegafur, a masked compound of 5-FU, and leucovorin in gastric cancer. The purpose of this study was to determine whether orally administered low-dose leucovorin enhances thymidylate synthase (TS) inhibition when added to tegafur-uracil (UFT) in patients with gastric cancer. METHODS: A group of 26 patients with resectable gastric cancer were assigned to one of two regimens: UFT alone or UFT plus leucovorin. UFT, equivalent to 400 mg/day tegafur, with or without 30 mg/day leucovorin, was administered orally in divided daily doses every 12 h for 3 consecutive days prior to surgery. Tumor specimens were taken immediately following gastrectomy, and the TS inhibition rate (TSIR) was determined using a ligand-binding assay. RESULTS: The TSIR was significantly higher in the UFT plus leucovorin group than in the UFT alone group (P < 0.01). The TSIR in the patients treated with UFT alone ranged between 14% and 50%, while six of the eight patients treated with UFT plus leucovorin had a TSIR of 55% or higher. The remaining two patients in the group treated with UFT plus leucovorin, with a TSIR of 31% and 44%, had undifferentiated tumors. CONCLUSION: Our results suggest that orally administered low-dose leucovorin can add to the efficacy of UFT in patients with gastric cancer, and provide preliminary data for a randomized clinical trial.

Surgical strategy for patients with gastric carcinoma with submucosal invasion. A multivariate analysis.

BACKGROUND: Early gastric cancer can be treated by endoscopic excision or simple wedge surgical resection. Standard gastrectomy often is advised if submucosal invasion is found, even though only 15-25% of these patients have lymph node metastases. In this study, the risk of lymph node involvement was examined by multivariate analysis to develop a simple discriminant function for surgical decision making in this setting. METHODS: The authors determined factors significantly correlated with lymph node involvement in a retrospective review of 196 patients with gastric adenocarcinoma invading into, but not beyond, the submucosa. Depth and horizontal spread of cancer in the submucosa were evaluated in addition to ordinary pathologic factors. Discriminant analysis for lymph node involvement was performed using explanatory variables chosen from the results of the univariate analyses. RESULTS: Lymph node involvement correlated significantly with larger tumor size; greater dimension of submucosal invasion; deeper submucosal invasion; gross appearance of Type I, IIc + III or IIa + IIc; and severity of vessel invasion. Of the variables, the amount of lymphatic invasion, macroscopic appearance, and maximum dimension of submucosal infiltration were selected as effective predictors of lymph node involvement according to discriminant analysis. A correct discrimination of 74.8% was obtained with a linear discriminant function using these variables. Lymph node involvement was observed in 50% of the cases with a discriminant score less than -1 and in 25% of those with a score between -1 and 0, whereas no lymph node involvement was observed in those with a score greater than 2. CONCLUSIONS: Discriminant function as used in this study provided a useful criterion for additional surgery for patients with gastric carcinoma invading the submucosa who were treated initially by localized excision. Prophylactic lymph node dissection may not be necessary for a discriminant score greater than 2, whereas extended lymphadenectomy would be recommended for a score less than -1.

Intron length variation of the Adh gene in Brachyscome (Asteraceae).

Eighteen polymerase chain reaction (PCR) products of the partial sequence of the Adh (alcohol dehydrogenase) gene from 10 Brachyscome species were sequenced and compared. These products contained the 5' three fourths of exon 4 and whole sequences of intron 3. They varied extensively in length due to the differences in length of intron 3. A total of 10 long insertions were flanked by direct repeats of 5 to 12 bp sequences, indicating inserted elements. These inserted elements were classified into the following five categories based on nucleotide sequence characteristics and length; (1) a region homologous to that of 5S RNA genes (5S DNA), (2) A-rich structure at the 3' end-like short interspersed elements (SINEs) in animals, (3) a sequence of 280 bp with no characteristic features, (4) a sequence of 125 bp with no characteristic features, (5) termini of 11 bp inverted repeats flanked by 5 bp sequence of direct repeats characteristics of a transposon.