A glycine-rich RNA-binding protein affects gibberellin biosynthesis in Arabidopsis.

The RNA-binding protein Arabidopsis thaliana glycine-rich RNA-binding protein 7 (AtGRP7) regulates the steady-state abundance of numerous target transcripts in A. thaliana. Here we show that the GA1 and GA2 transcripts encoding the first enzymes of the gibberellin biosynthetic pathway are expressed at reduced levels in transgenic plants ectopically over-expressing AtGRP7 (AtGRP7-ox plants). Furthermore, the levels of the bioactive phytohormone GA4 as well as of several intermediates of the GA biosynthetic pathway are reduced in AtGRP7-ox plants. The transgenic plants show a reduced length of the vegetative stem. The application of exogenous GA largely reverses the phenotype by increasing the number of vegetative internodes. AtGRP7-ox plants flower with fewer leaves than wt plants, suggesting that the floral promotive effect of AtGRP7 bypasses the effect of a reduced GA level in AtGRP7-ox plants. Upon GA treatment, AtGRP7-ox plants flower only slightly earlier than wild type plants. Thus, exogenous GA has only a small additional effect in reducing the number of leaves at the onset of flowering in AtGRP7-ox plants.

Developmental and UV Light Regulation of the Snapdragon Chalcone Synthase Promoter.

Expression directed by the 1.1-kb snapdragon chalcone synthase (CHS) promoter linked to the [beta]-glucuronidase reporter gene has been studied in transgenic tobacco. The pattern of expression of the chimeric gene was compared with the expression of the endogenous CHS genes in tobacco and snapdragon. We demonstrate that expression of the CHS promoter is controlled in both an organ-specific and tissue-specific manner. The highest level of expression was observed in immature seeds. Deletions were used to define regions of the promoter required for expression in roots, stems, leaves, seeds, and flower petals of transgenic plants. We have defined the minimal sequences required for expression in different organs and mapped regions of the promoter that influence expression in either a positive or negative manner. A promoter fragment truncated to -39 activates transcription in roots of 4-week-old seedlings, whereas a fragment extending to -197 bp directs expression in petals and seeds. A positive regulatory element located between -661 and -566 and comprising a 47-bp direct repeat is active in all tissues investigated except petals. UV light-regulated expression in leaves of transgenic tobacco seedlings is dependent on the presence of sequences also required for leaf-specific expression. Within the intact promoter, sequences that individually confer different patterns of expression interact to produce the highly regulated expression pattern of CHS.

Health insurance and female labor supply in Taiwan.

We examine whether the availability of subsidized health insurance to the non-working population in Taiwan affected the labor force participation of married women. Our empirical identification exploits the fact that such insurance was first made available to wives of government employees, before being made universally available in Taiwan in 1995. We find that the availability of insurance for non-workers was associated with a 4 percentage point decline in labor force participation among married women, with larger declines among women from low income households. Countries considering universal health insurance as in Taiwan should anticipate similar declines in labor force participation.

Quantitative determination of cephalexin in cephradine by NMR spectroscopy.

An NMR method to determine quantitatively the presence of cephalexin in cephradine was developed. The method is applicable to the chemical itself as well as to capsules and oral suspension formulations. The determination is based on the NMR signal arising from the five aromatic protons of the cephalexin molecule. Integration of this signal relative to a signal from cephradine provides the data necessary to determine the percentage of cephalexin present. The precision at the 2% cephalexin levels is +/- 0.18%. The time required to carry out a single analysis is about 10 min, and five analyses can be done in about 0.5 hr.

Quantitative NMR analysis of a four-component mixture of phenylglycine derivatives.

A rapid, accurate, and precise NMR analytical method for the analysis of phenylglycine, dihydrophenylglycine, tetrahydrophenylglycine, and cyclohexylglycine in combination with each other was developed. The method is based on the integration of the NMR signal characteristic of each component relative to the signal from tetramethylammonium bromide, which is added as an internal standard. No prior separation of the four components is required.

NMR spectroscopic analysis of 2-mercapto-5-methyl-1,3,4-thiadiazole in cefazolin.

A rapid, sensitive, and accurate method for the quantitative analysis of 2-mercapto-5-methyl-1,3,4-thiadiazole in cefazolin is presented. The method utilizes NMR spectroscopy and is based on the difference in the chemical shift of the methyl protons on free thiadiazole and the thiadiazole moiety of cefazolin.

The circadian system of Arabidopsis thaliana: forward and reverse genetic approaches.

It is now widely accepted that autoregulatory circuits involving transcription/translation of clock genes form the molecular basis of the endogenous circadian clock in different organisms. In Arabidopsis thaliana, the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich protein) has been identified as part of a negative-feedback loop through which AtGRP7 regulates the circadian oscillations of its own transcript. Experimental evidence indicates that this feedback loop also is influenced by another oscillator. Support for this hypothesis comes from the characterization of the clock mutant toc1 (timing of cab expression) and the recent isolation of two candidate clock molecules, LHY (late elongated hypocotyl) and CCA1 (circadian clock associated). TOC1, as well as the LHY and CCA1 oscillatory feedback loops, influence several rhythmic physiological and molecular processes in Arabidopsis, including cyclic Atgrp7 gene expression. We discuss the features of these feedback loops with relation to the organization of the circadian system in Arabidopsis.

Kibdelins (AAD-609), novel glycopeptide antibiotics. II. Isolation, purification and structure.

A new glycopeptide antibiotic complex was isolated from the fermentation culture of Kibdelosporangium aridum subsp. largum (SK&F AAD-609) by affinity chromatography on a D-alanyl-D-alanine agarose column. This major components of the complex were resolved by preparative reversed-phase HPLC. Mild acid hydrolysis showed that the new antibiotics have the same mannosyl aglycon (2) as the aridicins. FAB mass spectrometry, isoelectric focusing, potentiometric titration and carbohydrate and fatty acid analyses were used to determine the structures of the five major components of the complex. These studies showed that the kibdelins differ from the aridicins only in the oxidation level at the C-6 position of the amino sugar. Kibdelin A (5), B (6), C1 (7), C2 (8) and D (9) are a series of N-acylglucosamine analogs containing saturated straight and branched chain C10-C12 fatty acids whereas, in kibdelin D the fatty acid component is (Z)-4-decenoic acid.

Isolation and structure determination of Pachybasium cerebrosides which potentiate the antifungal activity of aculeacin.

A set of four cerebrosides was isolated from a Pachybasium species and purified by preparative reversed-phase HPLC. All four products displayed activity in a natural product screen aimed at detecting novel cell wall-active antifungal agents based on synergy with the known glucan synthetase inhibitor, aculeacin. Based on degradation studies, fast atom bombardment mass spectrometry and 13C and high field 1H NMR techniques, the structure of the major cerebroside was determined to be (4E,8E)-N-D-2'-hydroxy-(E)-3'- hexadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4,8-sphingadiene. The other components were found to be the corresponding 2'-hydroxypalmitic acid analog with one less double bond and an analogous pair containing 2'-hydroxystearic acid with and without the 3' double bond.

Associate degree graduates and the rapidly aging RN workforce.

This second segment of a four-part series examines the inter-relationship between the growth in associate degree nursing programs and the aging of the RN workforce. A growing proportion of new RNs have entered the workforce via associate degree programs, increasing from 40% in 1977 to 60% in 1996. New graduates, as well as working RNs, are approximately 5 years older in 1996 than 20 years earlier. Findings suggest that the rapid aging of the RN workforce can not be directly attributed to the rise in the number of older-aged graduates of associate degree programs. Rather, the declining propensity of those born after 1960 to enter nursing has resulted in fewer young RNs, and therefore: (1) an aging workforce, and (2) fewer new grads from baccalaureate programs (which have always attracted younger RNs) relative to grads from associate degree programs (which have always attracted older RNs).

Why are shortages of hospital RNs concentrated in specialty care units?

This article is the first in a series examining the interplay between the aging of the nurse workforce and other factors driving the growing nursing shortage that are already affecting some specialty areas. Nearly 60% of the current RN workforce is over 40 years of age; and the percentage of RNs under age 30 has fallen by nearly 40% since 1980. The total number of FTE RNs is projected to shrink after 2010, likely resulting in shortages of RNs "when the large baby-boom generation of RNs starts to retire." Because ICUs have historically attracted younger RNs, the rapid decline in the number of RNs in the workforce under age 30 plays a large role in explaining the development of shortages in the ICU. The growing difficulties staffing operating rooms and other peri-operative services is seen as related to the aging work force as more diploma prepared nurses have been attracted to this specialty because they had educational exposure to this area.

How hospital ownership affects access to care for the uninsured.

This article addresses the effect of hospital ownership on the delivery of service to uninsured patients. It compares the volume of uninsured patients treated in for-profit and nonprofit hospitals by regarding hospital ownership and service as endogenous. Instrumental variable estimates are used to predict the percentage of patients who are uninsured, controlling for hospital ownership and service. The study shows that when for-profit and nonprofit hospitals are located in the same area, they serve an equivalent number of uninsured patients, but for-profit hospitals indirectly avoid the uninsured by locating more often in better-insured areas.

RNA-binding proteins and circadian rhythms in Arabidopsis thaliana.

An Arabidopsis transcript preferentially expressed at the end of the daily light period codes for the RNA-binding protein AtGRP7. A reverse genetic approach in Arabidopsis thaliana has revealed its role in the generation of circadian rhythmicity: AtGRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit: Atgrp7 gene transcription is rhythmically activated by the circadian clock during the day. The AtGPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post-transcriptional level. In this respect, the AtGRP7 feedback loop differs from known circadian oscillators in the fruitfly Drosophila and mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that the AtGRP7 feedback loop may act within an output pathway from the Arabidopsis clock.

Circadian clock-regulated expression of an RNA-binding protein in Arabidopsis: characterisation of a minimal promoter element.

The Atgrp7 transcript encodes a clock-regulated, glycine-rich, RNA-binding protein in Arabidopsis thaliana and shows a circadian variation in steady-state abundance. Constitutive overexpression of its product, AtGRP7, in transgenic Arabidopsis plants depresses the oscillations of the endogenous Atgrp7 transcript, indicating that both the transcript and the protein are part of a clock-regulated negative feedback circuit. Here we characterise the upstream region of the Atgrp7 gene in order to begin to dissect the molecular basis of this oscillating autoregulatory feedback loop. Fusion of a 1.5-kb promoter fragment to the beta-glucuronidase (gus) reporter gene leads to circadian oscillations in the level of the gus transcript in transgenic Arabidopsis plants, with highest levels in the evening, indicating that transcription of the Atgrp7 gene is rhythmically activated by the endogenous circadian clock. A 265-bp fragment upstream of the transcription start site is necessary for high-amplitude Atgrp7 cycling. Within this region, a 56-bp clock-responsive element that confers a low-amplitude circadian oscillation (approximately threefold) with peak abundance in the early evening maps between positions -112 and -57. Another element necessary for augmenting the amplitude of the oscillation lies between -178 and -264. Genetic crosses between a line bearing a promoter-gus fusion and plants that overexpress AtGRP7 show that the promoter by itself does not mediate the negative feedback of AtGRP7 on the oscillations of its own transcript.

Molecular characterization of two cDNAs from Sinapis alba L. expressed specifically at an early stage of tapetum development.

Flower formation in the long-day plant Sinapis alba is strictly dependent on an inductive light treatment. Differential screening of an apex cDNA library prepared 10 days after flower induction against cDNAs from vegetative apices has identified two cDNA clones, pSFD10.35 and pSFD10.44, which represent transcripts expressed transiently between day 10 and day 20 after induction and which disappear before flowers reach maturity. The corresponding full-length cDNAs which were isolated, Satap35 and Satap44, display 86% overall sequence identity. These cDNAs encode polypeptides with predicted molecular weights of 12.7 and 12.4 kDa, and isoelectric points of 10.4 and 7.5, respectively. The N-terminal portions of the open reading frames have characteristics of signal sequences. In situ hybridization reveals that both transcripts are localized exclusively in the tapetal cell layer of the anthers. Maximal expression is observed in flower buds of approximately 1.5 mm length (tetrad stage). Southern blot analysis demonstrates the presence of additional, closely related genes in the Sinapis genome, as well as the occurrence of homologous genes in Arabidopsis thaliana, Brassica napus and Nicotiana tabacum.

The Atger3 promoter confers circadian clock-regulated transcription with peak expression at the beginning of the night.

In Arabidopsis thaliana, steady-state abundance of the Atger3 transcript encoding a germin-like cell wall protein follows a circadian rhythm, reaching its highest level at the beginning of the night. As a first step towards dissecting the molecular mechanisms underlying these transcript oscillations, the Atger3 genomic locus was characterised. Transcriptional fusions of 1.8 kb and 967 bp Atger3 promoter fragments to the beta-glucuronidase (GUS) reporter gene mediate high-amplitude circadian oscillations of the GUS transcript in transgenic Arabidopsis. 5' deletion to -490 greatly reduces overall transcript abundance while retaining a basal oscillation. Further deletion to -299 abolishes preferential GUS expression in the evening. Taken together, these data indicate that clock-response elements contributing to high-amplitude Atger3 oscillations largely reside between -299 and -967. Histochemical staining for GUS activity indicates that the Atger3 promoter is active in cotyledons, young leaves, petioles, the inflorescence axis, pedicels, sepals, ovary, style and siliques but not in roots, petals and anthers.

A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein.

In the chalcone synthase gene of Antirrhinum majus (snapdragon), 150 base pairs of the 5' flanking region contain cis-acting signals for UV light-induced expression. A nuclear factor, designated CG-1, specifically recognizes a hexameric motif with internal dyad symmetry, CACGTG, located within this light-responsive sequence. Binding of CG-1 is influenced by C-methylation of the CpG dinucleotide in the recognition sequence. CG-1 is a factor found in a variety of dicotyledonous plant species including Nicotiana tabacum, A. majus, Petunia hybrida, Arabidopsis thaliana, and Glycine max. CACGTG motifs contained within trans-acting factor recognition sites in various other plant promoters can interact with CG-1. In addition, the binding site of the human adenovirus major late transcription factor USF can compete for CG-1 binding to the chalcone synthase promoter. This suggests an evolutionary conservation of trans-acting factor recognition sites involved in divergent mechanisms of gene control.