Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene.

Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid alpha-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, alpha-linolenic acid content of the T(1) seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T(2) and T(3) progenies. These results demonstrate that the alpha-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.

Risk of irregular menstrual cycles and low peak bone mass during early adulthood associated with age at menarche.

BACKGROUND: The risk of irregular menstrual cycles and low peak bone mass of the lumbar spine in young adult women, associated with age at menarche, has not been clearly defined. The aim of this study was to investigate this further. METHODS: A total of 180 college females aged 20-23 years were surveyed about menstruation, exercise, and calcium intake by means of a questionnaire. We also measured vertebral bone mineral density in these women by dual energy X-ray absorptiometry. The subjects were divided into six groups according to their age at menarche. Comparisons were made of the mean body mass index (BMI) and T-scores (the ratio to the mean bone mineral density of young adult Japanese women) among those groups, and odds ratios of irregular menstrual cycles and low peak bone mass less than 87% (- 1 standard deviation (SD) of T-scores were calculated using the mean menarcheal age (12 years) group as a control. RESULTS: The mean BMI and T-scores were significantly lower in delayed menarche groups (equal to or more than 13 years) compared with early menarche groups (equal to or less than 12 years). The odds ratios of irregular menstrual cycles were 5.9 (95% confidence intervals (CI) 1.7-20.6), 13.7 (95% CI 3.6-51.6), and 73 (95% CI 6.5-813.9) in the 13-, 14-, and more than 14 years menarcheal age groups, respectively. The odds ratio of low peak bone mass less than 87% (- 1SD) was 3.4 (95% CI 1.1-10.3) in the 14 years menarcheal age group. CONCLUSIONS: Young adult women with delayed menarche may be at high risk for irregular menstrual cycles and low peak bone mass.

Gene transfer into hepatoma cells mediated by galactose-modified alpha-helical peptides.

To develop a receptor-mediated gene delivery system into hepatoma cells using the cationic alpha-helical peptide as the gene carrier molecule, we modified an alpha-helical peptide, which is known to have transfection abilities into cells, with a multi-antennary ligand containing several galactose residues that provide efficient binding to the asialoglycoprotein receptor. The galactose-modified peptides formed complexes with a plasmid DNA and showed gene transfer abilities into HuH-7 cells, a human hepatoma cell line. The transfection efficiency of the peptide was increased by increasing the number of modified galactose residues on the peptide. Furthermore, considerable inhibition of the transfection efficiency by the addition of asialofetuin, which is a ligand for the asialoglycoprotein receptor, was observed in all galactose-modified peptides. Based on this result, we could confirm that the internalization of the galactose-modified peptides occurred by the receptor-mediated endocytosis pathway. In addition, to understand the transport route of the peptide-DNA complex in the cell, the effects on the transfection efficiencies with several endocytosis inhibitors were examined. As a result, it was suggested that the translocation of the peptide-DNA complex from the endocytic compartments to the cytosol mainly occurred during an early endosome step.

A pair of invertedly repeated genes in Chlamydomonas reinhardtii encodes a zygote-specific protein whose expression is UV-sensitive.

Uniparental inheritance of the chloroplast genome has been observed in a wide variety of green plants. In Chlamydomonas this phenomenon, which can be selectively inhibited by UV irradiation of mt(+) gametes, has been shown cytologically to be due to the preferential degradation of mt(-)-derived chloroplast nucleoids in young zygotes. The zygote-specific pair of zys1 genes, zys1A and zys1B, is expressed earliest among five genes isolated from a "10-min" zygote library. We report here that the ZYS1 protein, which is encoded by the invertedly duplicated zys1 gene, accumulates in zygotes and is localized in nuclei. In addition, when mt(+) gametes (but not mt(-) gametes) are UV-irradiated before mating, only very limited accumulation of ZYS1 protein can be detected in the resulting zygotes.

Remission of idiopathic hypoparathyroidism during lactation: a case report.

A 35-year-old woman with idiopathic hypoparathyroidism achieved a spontaneous remission during lactation even though 1-alpha-hydroxyvitamin D3 therapy was discontinued after delivery. Urinary excretion of cyclic adenosine 3',5'-monophosphate and phosphate was significantly increased during lactation, probably in response to the increased levels of circulating parathyroid hormone-related protein derived from the breast tissue.

Pregnancy-associated osteoporosis with elevated levels of circulating parathyroid hormone-related protein: a report of two cases.

Two lactating women who had complained of back pain developed spontaneous vertebral fractures with low bone mineral density (BMD) several months postpartum. The back pain and biochemical abnormalities presented as hypercalcemia and elevated plasma levels of the parathyroid hormone-related protein (PTH-rP) that returned to normal indices with increasing BMD after weaning. The increased circulating PTH-rP might contribute to the pregnancy-associated osteoporosis in women who probably are already osteopenic.

Interleukin-1beta and interleukin-8 in cervicovaginal fluid during pregnancy.

OBJECTIVE: We investigated the usefulness of cervicovaginal interleukin-1beta and interleukin-8 levels during pregnancy as markers of preterm and term parturition. STUDY DESIGN: Cervicovaginal fluids were obtained from 119 pregnant women at 24 to 41 weeks' gestation. Cytokine concentrations were measured by enzyme-linked immunoassay. RESULTS: Interleukin-1beta and interleukin-8 concentrations in cervicovaginal fluids obtained from women not in labor increased exponentially as gestational age increased. Interleukin-1beta and interleukin-8 concentrations were significantly correlated. These cytokine concentrations were significantly higher in patients in preterm labor than in patients at preterm not in labor and were significantly higher in women at term in labor than in women at term not in labor. These cytokine levels were significantly correlated with the degree of cervical dilation and tocolytic index. CONCLUSIONS: These findings suggest that cervicovaginal interleukin-1beta and interleukin-8 contribute to term and preterm parturition. Measurement of cervicovaginal concentrations of these cytokines may be clinically useful in patients with threatened premature delivery.

Magnetic resonance imaging of fetal autosomal recessive polycystic kidney disease.

We present a case of fetal autosomal recessive polycystic kidney disease (ARPKD) at 34 weeks of gestation, diagnosed by magnetic resonance imaging (MRI). MRI demonstrated enlarged fetal kidneys that were low signal intensity on T1-weighted images and high signal intensity on T2-weighted images. These MRI findings suggested a high water content in the renal parenchyma, consistent with the innumerable, tiny renal cysts present in ARPKD.

AtGDI2, a novel Arabidopsis gene encoding a Rab GDP dissociation inhibitor.

The GTPase cycle of Rab/Ypt proteins is strictly controlled by several classes of regulators to ensure their proper roles in membrane traffic. GDP dissociation inhibitor (GDI) is known to play essential roles in regulating nucleotide states and subcellular localizations of Rab/Ypt proteins. To obtain further knowledge on this regulator molecule in plants, we isolated and characterized two genes of Arabidopsis thaliana that encode different GDIs. AtGDI1 has been identified by a novel functional cloning in yeast [Ueda et al. (1996) Plant Cell, 8, 2079-2091] and AtGDI2 was isolated by cross-hybridization in this study. AtGDI2, as well as AtGDI1, complements the yeast sec19/gdi1 mutant, indicating that they can replace the function of yeast GDI. Evidence is shown that both AtGDI1 and AtGDI2 can interact with Ara4, an Arabidopsis Rab protein, in the yeast ypt1 mutant cells. AtGDI2 is ubiquitously expressed in Arabidopsis tissues with some difference from AtGDI1 in expression level. Genomic DNA hybridization using specific probes reveals the presence of one more GDI gene in Arabidopsis. This may imply differentiated roles of GDI in higher plants.

Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones.

Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking. The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome. Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70-90kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995. Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J. 7, 351-358), we have now established such a set of framework clones. To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs). In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes. This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes. Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap.

Nucleotide sequence of oat (Avena sativa L.) cDNA encoding an auxin-binding protein (ABP1).

We isolated and determined a nucleotide sequence of a cDNA clone encoding a protein homologous to maize major auxin-binding protein (ABP1) from a cDNA library of oat coleoptiles. The deduced amino acid sequence of this clone contained an N-linked glycosylation signal and an ER-retention signal. Furthermore, two domains that were important to interact with auxins, were conserved in this clone at amino acid level.

Isolation and characterization of an auxin-inducible SAUR gene from radish seedlings.

A gene homologue to the auxin-inducible SAUR (Small Auxin Up RNAs) of soybean was isolated from a cDNA and a genomic library of radish. The cDNA clone was about 470 bp in length and contained a DST (Down STream) element that was involved in mRNA instability in the 3'-untranslated region. The genomic clone contained two short sequences that related to auxin-responsible core sequences (AGTCTC and TATCCCAC) in its promoter region. The radish SAUR transcript was accumulated by auxin and cycloheximide treatments in the same way as that in other plant species. However, the NDE and DUE elements that were reported in all of the other SAUR gene promoters were not conserved in this radish SAUR gene. The results suggest that the promoter activity of the SAUR gene will be regulated by only two short sequences.

Parathyroid hormone-related protein levels in maternal and cord blood.

OBJECTIVE: Our purpose was to determine the plasma levels of parathyroid hormone-related protein in normal pregnant women in each trimester, at 1 month post partum in women using different feeding methods, and in umbilical venous blood. STUDY DESIGN: We studied 40 healthy nonpregnant women, 90 healthy pregnant women (30 in the each trimester), and 140 postpartum women (breast-feeding 74, mixed feeding 33, bottle feeding 33). We also measured the parathyroid hormone-related protein level in umbilical venous blood in 24 women. RESULTS: The means and SDs of the plasma parathyroid hormone-related protein level were 1.04 +/- 0.11 pmol/L (0.81 to 1.26 pmol/L) in nonpregnant women, 1.06 +/- 0.19 pmol/L (0.69 to 1.48 pmol/L) in pregnant women in the first trimester, 1.10 +/- 0.19 pmol/L (0.80 to 1.50 pmol/L) in pregnant women in the second trimester, and 1.17 +/- 0.16 pmol/L (0.86 to 1.57 pmol/L) in pregnant women in the third trimester. At postpartum month 1 the plasma level of parathyroid hormone-related protein was 1.25 +/- 0.23 pmol/L (0.83 to 2.01 pmol/L) in breast-feeding women, 1.17 +/- 0.16 pmol/L (0.84 to 1.53 pmol/L) in mixed-feeding women, and 1.03 +/- 0.16 pmol/L (0.74 to 1.43 pmol/L) in bottle-feeding women. The umbilical venous blood level of parathyroid hormone-related protein was 1.33 +/- 0.32 pmol/L (0.84 to 2.11 pmol/L). The mean plasma level of parathyroid hormone-related protein increased throughout pregnancy and was significantly high in the third trimester and closely associated with the degree of breast-feeding at 1 month post partum. The parathyroid hormone-related protein level in umbilical venous blood was significantly higher than those in any groups of pregnant women. CONCLUSIONS: Parathyroid hormone-related protein produced in the fetoplacental unit, the breast, or both can reach the maternal circulation.

Vaginal fluid hCG levels for detecting premature rupture of membranes.

OBJECTIVE: To determine if the measurement of hCG levels in vaginal fluid is useful for the diagnosis of premature rupture of membranes (PROM). METHODS: After irrigating the posterior vaginal fornix with 3 mL of sterile saline and procuring vaginal washings, we measured hCG levels. Samples were analyzed from 188 normal pregnant women, 42, 61, and 85 during the first, second, and third trimesters, respectively. Levels of hCG were compared with those of 24 women with confirmed PROM. RESULTS: The median and 95% confidence intervals (CIs) of vaginal fluid hCG levels of normal pregnant women were 37.9 (1.9, 725.6), 9.5 (0.8, 95.8), and 6.3 (0.6, 62.2) mIU/mL during the first, second, and third trimesters, respectively. That of women with PROM was 420.6 (216.3, 918.3) mIU/mL. For the second trimester, sensitivity was 100%, specificity 91.8%, positive predictive value 82.8%, negative predictive value 100%, and accuracy 94.1%; and for the third trimester, sensitivity was 100%, specificity 96.5%, positive predictive value 88.9%, negative predictive value 100%, and accuracy 97.2%, using a threshold value of 50 mIU/mL. CONCLUSION: The hCG level in vaginal fluid is a useful marker of PROM during the second and third trimesters.

An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.

The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.

Characterization and subcellular localization of a small GTP-binding protein (Ara-4) from Arabidopsis: conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1.

Small GTP-binding proteins belonging to the rab/YPT family play key roles at various steps in intracellular transport pathways in yeast and mammalian cells. Many members of rab/YPT family have been isolated from plants to date. However, detailed information about the localization and function of the gene products remains limited, even though intracellular transport is likely to be involved in important phenomena such as cell elongation, transport of storage proteins, determination and maintenance of cell polarity and intercellular signal transduction. We have attempted to establish transgenic Arabidopsis plants that overexpress ARA-4, a rab/YPT homologue in order to analyze the function and the localization of the gene product. For overexpression and also for regulation of the expression of this gene, the promoter of the gene for HSP81-1 was employed to drive the transcription of ARA-4 in transgenic plants. The response of the introduced genes to heat shock was analyzed. Upon heat-shock treatment, the ARA-4 gene was efficiently transcribed and translated. The induction of ARA-4 by heat shock was transient, and at least two distinct forms of this protein were found in membrane and cytosolic fractions from transgenic plants. Prolonged incubation after heat shock reduced the amount of the cytosolic form of the induced protein, and the cytosolic form of the protein thus probably represents the unprocessed precursor. Using transgenic plants, we determined the subcellular localization of the product of ARA-4. The protein was predominantly localized on Golgi-derived vesicles, Golgi cisternae and the trans-Golgi network.

Structure-activity relationship of lepidimoide and its analogues.

The structure-activity relationship of lepidimoide and its analogues was investigated by means of the Amaranthus caudatus L. hypocotyl elongation test. In addition, the activities of alpha-D-galacturonic acid and L-(+)-rhamnose, which are component sugars of lepidimoide, were also studied. The carboxylic acid free type of lepidimoide showed growth-promoting activity as high as the original lepidimoide (sodium type). The acetylated compound showed considerably higher activity than lepidimoide, whereas the methylated lepidimoide did not show any activity. The hydroxylated lepidimoide without a double bond in the C-4,5 position showed lower activity. The sugar alcohol type of lepidimoide [2-O-(alpha-D-glucopyranosyl)-L-rhamnose] showed the highest activity in all the compounds studied. alpha-D-Galacturonic acid, L-(+)-rhamnose and their mixtures, which are component sugars of lepidimoide, exhibited only slight or no activity, respectively. D-Glucose and the mixture of D-glucose and L-(+)-rhamnose were also slightly active or inactive. These data suggest that the active sites in the chemical structure of the lepidimoide are the uronic acid derivative bearing an alpha,beta-unsaturated carboxylate bonded to rhamnose via an alpha-glucoside linkage and a double bond in the C-4,5 position in the uronic acid.

Novel DNA probes capable of discriminating indica and japonica rice cultivars.

We obtained two DNA clones, G93 (1.6kb) and G318 (1.7kb) from indica rice (Oryza sativa L.) cultivar Culture 340. Southern hybridization analysis indicated that both DNA probes hybridized with DNA of indica cultivars but not with japonica cultivars. Some javanica cultivars DNAs did not hybridize with these probes. Nucleotide sequence (400bp) of a part of G93 showed a 65.8% G + C content. These novel DNA probes can be used for the study of evolutional fingerprinting of rice cultivars.

Low-dose dopamine in treating acute renal failure caused by preeclampsia.

Acute renal failure is a serious complication of preeclampsia that usually requires the termination of pregnancy. We present a case of acute renal failure due to severe preeclampsia successfully treated with the infusion of a low dose of dopamine. This 25-year-old Japanese primigravida was admitted at 31 weeks of gestation for the treatment of preeclampsia. Urine output was decreased to 380 ml/day; 24-hour creatinine clearance was decreased to 13.7 liters/day. Blood urea nitrogen was elevated to 31.9 mg/dl; serum creatinine was elevated to 3.34 mg/dl. The diagnosis was acute renal failure related to preeclampsia. A low dose of dopamine, 1 microgram/kg/min, was infused daily for 7 days at 32 weeks of gestation to maintain urine output. Renal function improved markedly without any adverse effect on the patient's blood pressure which was controlled on hydralazine. Fetal distress developed 4 days later and emergency cesarean section was performed. A healthy female was delivered. The infusion of a low dose of dopamine appeared to be highly effective in managing acute renal failure caused by preeclampsia with no serious side effects.

Vertebral bone mineral density in postpartum women.

The purpose of this study was to measure the bone mineral density of the lumbar spine (L2-L4) in healthy postpartum women following term delivery, and to evaluate factors associated with bone mineral density. Using dual energy X-ray absorptiometry, the bone mineral density of the lumbar spine was determined during 1 year in 123 healthy postpartum Japanese women following term delivery. The mean bone mineral density of the lumbar spine was 1.007 +/- 0.108 g/cm2 (range 0.731-1.252 g/cm2). Thus, there were wide inter-individual differences. Also, this result was in agreement with the peak bone mass previously reported for other Japanese women. Multiple regression analysis and linear regression analysis showed that weight and body mass index were each significantly correlated with bone mineral density of the lumbar spine. Age, parity, height, and baby's birth weight were not contributory.