Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species.

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms-a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.

Medical injuries among hospitalized children.

BACKGROUND: Inpatient medical injuries among children are common and result in a longer stay in hospital and increased hospital charges. However, previous studies have used screening criteria that focus on inpatient occurrences only rather than on injuries that also occur in ambulatory or community settings leading to hospital admission. OBJECTIVE: To describe the incidence and outcomes of medical injuries among children hospitalized in Wisconsin using the Wisconsin Medical Injury Prevention Program (WMIPP) screening criteria. METHODS: Cross sectional analysis of discharge records of 318,785 children from 134 hospitals in Wisconsin between 2000 and 2002. RESULTS: The WMIPP criteria identified 3.4% of discharges as having one or more medical injuries: 1.5% due to medications, 1.3% to procedures, and 0.9% to devices, implants and grafts. After adjusting for the All Patient Refined-Diagnosis Related Groups disease category, illness severity, mortality risk, and clustering within hospitals, the mean length of stay (LOS) was a half day (12%) longer for patients with medical injuries than for those without injuries. The similarly adjusted mean total hospital charges were 1614 dollars (26%) higher for the group with medical injuries. Excess LOS and charges were greatest for injuries due to genitourinary devices/implants, vascular devices, and infections/inflammation after procedures. CONCLUSIONS: This study reinforces previous national findings up to 2000 using Wisconsin data to the end of 2002. The results suggest that hospitals and pediatricians should focus clinical improvement on medications, procedures, and devices frequently associated with medical injuries and use medical injury surveillance to track medical injury rates in children.

Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana.

An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimised for the development of protoplasts embedded in thin alginate layers. Unexpectedly, the absence of cytokinins had a positive effect on cell development. Moreover, combined adjustment of alpha-naphthylacetic acid and dicamba concentrations resulted in high plating efficiencies of up to 30%, followed by shoot regeneration within only 19 days after protoplast isolation. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana.

Effects of selective inactivation of individual genes for low-molecular-mass subunits on the assembly of photosystem II, as revealed by chloroplast transformation: the psbEFLJoperon in Nicotiana tabacum.

Photosystem (PSII) is a supramolecular polypeptide complex found in oxygenic photosynthetic membranes, which is capable of extracting electrons from water for the reduction of plastoquinone. An intriguing feature of this assembly is the fact that it includes more than a dozen low-mass polypeptides of generally unknown function. Using a transplastomic approach, we have individually disrupted the genes of the psbEFLJoperon in Nicotiana tabacum, which encode four such polypeptides, without impairing expression of downstream loci of the operon. All four mutants exhibited distinct phenotypes; none of them was capable of photoautotrophic growth. All mutants bleached rapidly in the light. Disruption of psbEand psbF, which code for the alpha and beta apoproteins of cytochrome b(559), abolished PSII activity, as expected; Delta psbL and Delta psbJ plants displayed residual PSII activity in young leaves. Controlled partial solubilisation of thylakoid membranes uncovered surprisingly severe impairment of PSII structure, with subunit and assembly patterns varying depending on the mutant considered. In the Delta psbL mutant PSII was assembled primarily in a monomeric form, the homodimeric form was preponderant in Delta psbJ, and, unlike the case in Delta psbZ, the thylakoids of both mutants released some PSII supercomplexes. On the other hand, Photosystem I (PSI), the cytochrome b(6)f complex, ATP synthase, LHCII, and CP24/CP26/CP29 antennae were present in near wild-type levels. The data are discussed in terms of their implications for structural, biogenetic and functional aspects of PSII.

Urban Wisconsin pediatric patients using an after-hours telephone triage service: outcomes and compliance.

OBJECTIVE: Evaluate the ability of a telephone triage service (TTS) to assess illness acuity of and patient compliance with advice given. DESIGN: Retrospective, observational study. PATIENTS: Patients of an urban, academic, pediatric clinic whose parents or caregivers called the TTS between July 23, 1997 and August 23, 1997. OUTCOME MEASURES: Patient outcomes and visit information at related medical encounters subsequent to a TTS call. RESULTS: Patients were primarily African-American, under age 5, enrolled in a Medicaid HMO, and most often called for fever, HMO authorization, or asthma. Homecare and PED referrals were the two most frequent dispositions; overall compliance rate was 60%. No patient referred for non-emergent care required care on an urgent or emergent basis. CONCLUSIONS: Initial results suggest that the TTS can effectively evaluate illness acuity in an urban population and compliance with advice is reasonable. A TTS may offer significant benefits to ensure care quality and contain costs in this population.

Deregulation of electron flow within photosystem II in the absence of the PsbJ protein.

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.

The nucleus-encoded HCF107 gene of Arabidopsis provides a link between intercistronic RNA processing and the accumulation of translation-competent psbH transcripts in chloroplasts.

To understand the functional significance of RNA processing for the expression of plastome-encoded photosynthesis genes, we investigated the nuclear mutation hcf107 of Arabidopsis. The mutation is represented by two alleles, both of which lead to a defective photosystem II (PSII). In vivo protein labeling, in vitro phosphorylation, and immunoblot experiments revealed that the psbB gene product (CP47) and an 8-kD phosphoprotein, the psbH gene product (PsbH), are absent in mutant plants. PsbH and PsbB are essential requirements for PSII assembly in photosynthetic eukaryotes, and their absence in hcf107 is consistent with the PSII-less mutant phenotype. RNA gel blot hybridizations showed that the hcf107 mutation specifically impairs the accumulation of some but not all oligocistronic psbH transcripts that are released from the pentacistronic psbB-psbT-psbH-petB-petD precursor RNA by intergenic endonucleolytic cleavage. In contrast, neither the levels nor the sizes of psbB-containing RNAs are affected. S1 nuclease protection analyses revealed that psbH RNAs are lacking only where psbH is the leading cistron and that they are processed at position -45 in the 5' leader segment of psbH. These data and additional experiments with the cytochrome b(6)f complex mutant hcf152, which is defective in 3' psbH processing, suggest that only those psbH-containing transcripts that are processed at their -45 5' ends can be translated. Secondary structure analysis of the 5' psbH leader predicted the formation of stable stem loops in the nonprocessed transcripts, which are unfolded by processing at the -45 site. We propose that this unfolding of the psbH leader segment as a result of RNA processing is essential for the translation of the psbH reading frame. We suggest further that HCF107 has dual functions: it is involved in intercistronic processing of the psbH 5' untranslated region or the stabilization of 5' processed psbH RNAs, and concomitantly, it is required for the synthesis of CP47.

Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis.

The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of T-DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutation responsible was localized to Prpl11, a single-copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes. In the prpl11-1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins. The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased. Plastid ribosomal RNA accumulates in wild-type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes. However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11-1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase-binding centre of the chloroplast 50S ribosomal subunit. Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.

Map positions of 69 Arabidopsis thaliana genes of all known nuclear encoded constituent polypeptides and various regulatory factors of the photosynthetic membrane: a case study.

Landsberg erecta x Columbia F8 recombinant inbred lines of Arabidopsis thaliana, arrayed BAC clones covering most of the genome, and databank sequence information were used to map the positions of 69 genes in the genome of A. thaliana. These genes encode all known constituents of the photosynthetic thylakoid membrane, some regulatory factors involved in its biogenesis, and the RNA polymerases of nuclear origin that operate in chloroplasts and mitochondria. Designations of novel genes are proposed. The data of these three approaches are generally consistent, although ambiguities have been noted for some genome segments and with gene duplications. For thylakoid multi-subunit structures, no positional clustering of genes has been found, not even for genes encoding different subunits of the same membrane complex. The genes of the lhc superfamily encoding antenna apoproteins and their relatives are a particularly intriguing example. The lack of positional clustering is consistent with phylogenetically independent gene translocations from the plastid (endosymbiont) to the nucleus. This raises the basic question of how independently translocated genes which acquired different promoter sequences and transit peptides were functionally integrated into common signal transduction chains.

Pediatric Asthma Study of the State Medical Society of Wisconsin.

PROBLEM: Asthma management should follow evidence-based national guidelines. We compared patient self-reports of usual care to clinical practice guidelines. METHODS: Cross-sectional survey of parents of 245 children age 2-14 years with asthma seen by physicians at 13 practices in Wisconsin. RESULTS: Most children with asthma were atopic and a substantial number experienced psychosocial problems. Families infrequently used written care plans. Knowledge about asthma triggers, especially tobacco smoke, was deficient. Increasing asthma severity was associated with poorer health. Although most parents rated their physician's expertise as excellent or very good, one-third of children with persistent symptoms used no daily control medication. Significant differences were observed between primary care and specialty care with the best asthma self-management done by patients of specialists. CONCLUSION: Care of Wisconsin children with asthma inconsistently follows national guidelines. They and their families require improved education, written care plans, appropriate medications, and psychosocial support.

Newborn hearing testing at Wisconsin hospitals: a review of the need for universal screening.

Every year, an estimated 200 babies are born in Wisconsin with hearing impairment. Early identification of hearing loss is associated with better language development. This study describes the prevalence of newborn hearing screening at Wisconsin hospitals. Ninety-six percent of all 107 acute care hospitals with newborn delivery services responded to a mailed survey. In 1997, 10% of newborns in Wisconsin had hearing tested. In 1997 and 1998, two and then seven hospitals, respectively, established universal hearing screening programs. Nurses performed hearing tests on most newborns using automated auditory brainstem response equipment. Universal newborn hearing screening can be done by nurses or other personnel at community hospitals prior to discharge.

Disruption of the Arabidopsis photosystem I gene psaE1 affects photosynthesis and impairs growth.

The psae1-1 mutant of Arabidopsis was identified on the basis of a decrease in the effective quantum yield of photosystem II, among a collection of plants subjected to transposon tagging with the Enhancer element. The steady-state redox level and the rate of re-oxidation of P700 are significantly altered in psae1-1 mutants. The responsible mutation was localised to psaE1, one of two Arabidopsis genes that encode subunit E of photosystem I. An additional mutant allele, psae1-2, was identified by reverse genetics. In wild-type plants, the psaE1 transcript is expressed at a higher level than psaE2 mRNA. In the mutants, however, the E1 transcript was barely detectable, and was expressed only in small groups of wild-type cells resulting from somatic reversions. As a consequence, the amount of PsaE protein present in the mutant is significantly reduced. Concomitantly, the levels of other stromal photosystem I subunits (PsaC and PsaD) are also affected. Mutant plants showed a marked increase in light sensitivity and photoinhibition. Additional effects of the psae1 mutation include light green pigmentation, an increase in chlorophyll fluorescence and a decrease of approximately 50% in growth rate under greenhouse conditions.

Asthma severity among children hospitalized in 1990 and 1995.

BACKGROUND: During the past decade, the number of children with asthma increased; however, the number of asthma hospitalizations for children decreased. OBJECTIVE: To assess the proportion of high-severity cases among children hospitalized with asthma and the association of high-severity asthma with patient and hospital characteristics. DESIGN: The data set used was the Healthcare Cost and Utilization Project Nationwide Inpatient Sample. Records were selected of patients 18 years and younger who had the principal diagnosis of asthma. Records were analyzed of 29077 patients at 746 hospitals in 1990 and 33 443 patients at 811 hospitals in 1995. Asthma severity was classified by All Patient Refined-Diagnosis-Related Groups. Cross-sectional logistic regression analysis was performed using survey data analysis software. RESULTS: The most common diagnoses associated with high-severity asthma were respiratory distress and respiratory failure. The proportion of high-severity asthma cases did not change significantly between 1990 (4.2%) and 1995 (4.6%) (P = .08). Adolescents and boys were more likely to have high-severity asthma than children aged 5 to 12 years and girls. Western, southern, and north-central hospitals and urban teaching hospitals had a higher proportion of high-severity asthma cases than northeastern hospitals and nonteaching hospitals. CONCLUSIONS: Between 1990 and 1995, the proportion of high-severity cases among children hospitalized with asthma did not change significantly. However, patient age, sex, region of the country, and hospital teaching status were associated with variations in the proportion of high-severity asthma cases.

Risk factors for pediatric asthma emergency visits. Milwaukee Childhood Asthma Project Team.

The purpose of this study was to identify risk factors for pediatric asthma emergency department (ED) visits. A cross-sectional sample of 307 families (24%) responded to a mailed survey. Respondents were similar to the target population of 2487 children with asthma. Thirty-seven percent of respondents had two or more asthma ED visits in the past year. Asthma ED use was associated with persistent asthma symptoms, parents without high school diplomas, age under 4 years, African-American race/ethnicity, use of written care plans, and use of spacers at school. The latter two variables were associated with severe asthma. High-risk children can be targeted for office-based medical care.

The assembly of the CAAT-box binding complex at a photosynthesis gene promoter is regulated by light, cytokinin, and the stage of the plastids.

A functionally important region in the promoter of the spinach photosynthesis gene AtpC, which encodes the subunit gamma of the chloroplast ATP synthase, is located immediately upstream of the CAAT-box. A single nucleotide exchange in this region (AAAATTCAAT --> AAGATCAAT) uncouples the expression of an AtpC promoter::uidA gene fusion from the regulation by light, cytokinin, and functional plastids and results in a high constitutive expression of the reporter gene. By screening an Arabidopsis thaliana expression library with a double-stranded wild-type oligonucleotide from this promoter region, we have isolated cDNAs from Arabidopsis libraries that code for plant homologs of the CAAT-box binding factor (CBF)-C. Binding occurs only in the presence of nuclear extracts, consistent with reports from metazoa CBFs that the subunits A and B in addition to C are required for the formation of the CBF-DNA complex. At least eight genes with homologies to CBF-C are present in the Arabidopsis genome; one of them exhibits striking similarities to the gene for the human global transcriptional repressor Drap1. In gel mobility shift assays, low binding activity of CBF to the wild-type AtpC promoter sequence was observed with nuclear extracts from tissue with low AtpC expression levels, i.e. extracts from etiolated and photobleached seedlings, whereas high binding activity was detectable with extracts from tissues with high AtpC expression levels, i.e. extracts from light-grown seedlings and etiolated seedlings treated with cytokinin. Binding to the mutant sequence, which directs constitutive high level uidA expression in vivo, is significantly stronger than to the wild-type sequence. The data are consistent with the idea that the assembly of CBF at the AtpC promoter is regulated in response to light and cytokinin and that the low level of expression in etiolated and photobleached material is caused by an inhibitory effect. The structure/function relationships of the Arabidopsis CBFs are discussed in relation to their regulatory function in AtpC gene expression.

Properties of native and in vitro glycosylated forms of the glucagon-like peptide-1 receptor antagonist exendin(9-39).

The intestinal hormone glucagon-like peptide-1-(7-36)-amide (GLP-1) has recently been implicated as a possible therapeutic agent for the management of type 2 non-insulin-dependent diabetes mellitus (NIDDM). However, a major difficulty with the delivery of peptide-based agents is their short plasma half-life, mainly due to rapid serum clearance and proteolytic degradation. Using a peptide analog of GLP-1, the GLP-1 receptor antagonist exendin(9-39), we investigated whether the conjugation of a carbohydrate structure to exendin(9-39) would generate a peptide with intact biological activity and improved survival in circulation. The C-terminal portion of exendin(9-39) was reengineered to generate an efficient site for enzymatic O-glycosylation. The modified exendin(9-39) peptide (exe-M) was glycosylated by recombinant UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) alone or in conjunction with a recombinant GalNAc alpha2,6-sialyltransferase (Sialyl-T), resulting in exe-M peptides containing either the monosaccharide GalNAc or the disaccharide NeuAc alpha2,6GalNAc. The nonglycosylated and glycosylated forms of exe-M competed with nearly equal potency (> 90% of control) with the binding of [125I]GLP-1 to human GLP-1 receptors expressed on stably transfected COS-7 cells. In addition, each peptide was equally effective for inhibiting GLP-1-induced cyclic adenosine monophosphate (cAMP) production in vitro. Studies with rats demonstrated that the modified and glycosylated forms of exendin(9-39) could antagonize exogenously administered GLP-1 in vivo. Interestingly, glycosylated exendin(9-39) homologs were more than twice as effective as the nonglycosylated peptide for inhibiting GLP-1-stimulated insulin production in vivo, suggesting a longer functional half-life in the circulation for glycosylated peptides. Results from in vivo studies with 3H-labeled peptides suggest that the glycosylated peptides may be less susceptible to modification in the circulation.

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis.

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.

The awesome Asthma School Days Program: educating children, inspiring a community.

Program planners developed an educational program to improve the health of children with asthma in grades three to five in Milwaukee (Wis.) Public Schools. During 1997-1998, 1,400 students from 74 elementary schools participated in the Awesome Asthma School Days education program. In a cross-sectional survey, about 40% of children reported play interrupted and sleep disturbed by asthma, more than 50% of children reported exposure to smoke in their home, most children lacked asthma self-care tools, and most children with persistent symptoms did not use an anti-inflammatory inhaler. The educational program improved students' expectations about normal play and sleep and improved their understanding of asthma. Leaders in Milwaukee used the survey results to develop a community action plan. The educational program, surveys, community partnerships, and strategic plans can be replicated in other schools.