Treatment of potato farm wastewater with sand filtration.

This study examined sand filtration as a component of a potato farm wastewater treatment system. Two different sand filter designs, saturated flow and unsaturated flow, were evaluated at three different loading rates: 34, 68, and 136 L m(-2) d(-1). Filter design had a significant effect, with unsaturated flow sand filters having significantly (p < .05) better total suspended solids (TSS) removal (89%) than saturated flow sand filters did (79%). Loading rate also had a significant (p < .05) effect, given that the lowest loading rate had higher mass removal for TSS than the higher loading rates did. Overall, all sand filters removed TSS, 5-d biochemical oxygen demand, and total phosphorus well (62-99%). Total nitrogen removal was twice as high in unsaturated flow filters (53%) than in saturated flow filters (27%), because of the recurring cycle of aerobic and anaerobic conditions during sand saturation and drying in unsaturated flow sand filters.

Hypothermia in very low birth weight infants: distribution, risk factors and outcomes.

OBJECTIVE: The objective of this study was to study the epidemiology of neonatal hypothermia in preterm infants using World Health Organization (WHO) temperature criteria. STUDY DESIGN: A population-based cohort of 8782 very low birth weight (VLBW) infants born in California neonatal intensive care units in 2006 and 2007. Associations between admission hypothermia and maternal and neonatal characteristics and outcomes were determined using logistic regression. RESULT: In all, 56.2% of infants were hypothermic. Low birth weight, cesarean delivery and a low Apgar score were associated with hypothermia. Spontaneous labor, prolonged rupture of membranes and antenatal steroid administration were associated with decreased risk of hypothermia. Moderate hypothermia was associated with higher risk of intraventricular hemorrhage (IVH). Moderate and severe hypothermic conditions were associated with risk of death. CONCLUSION: Hypothermia by WHO criteria is prevalent in VLBW infants and is associated with IVH and mortality. Use of WHO criteria could guide the need for quality improvement projects targeted toward the most vulnerable infants.

Hourglass cell development in the soybean seed coat.

BACKGROUND AND AIMS: Hourglass cells (HGCs) are prominent cells in the soybean seed coat, and have potential use as 'phytofactories' to produce specific proteins of interest. Previous studies have shown that HGCs initiate differentiation at about 9 d post-anthesis (dpa), assuming their characteristic morphology by 18 dpa. This study aims to document the structural changes in HGCs during this critical period, and to relate these changes to the concurrent development of a specific soybean peroxidase (SBP) encoded by the Ep gene. METHODS: Pods were collected from plants at specific growth stages. Fresh material was processed for analysis of Ep peroxidase activity. Tissues were processed for scanning and transmission electron microscopy, as well as extracted for western blotting. A null variety lacking expression of Ep peroxidase was grown as a control. KEY RESULTS AND CONCLUSIONS: At 9 dpa, HGCs are typical undifferentiated plant cells, but from 12-18 dpa they undergo rapid changes in their internal and external structure. By 18 dpa, they have assumed the characteristic hourglass shape with thick cell walls, intercellular air spaces and large central vacuoles. By 45 dpa, all organelles in HGCs have been degraded. Additional observations indicate that plasmodesmata connect all cell types. SBP activity and SBP protein are detectable in the HGC before they are fully differentiated (approx. 18 dpa). In very early stages, SBP activity appears localized in a vacuole as previously predicted. These results increase our understanding of the structure and development of the HGC and will be valuable for future studies aimed at protein targeting to components of the HGC endomembrane systems.

Transcriptome analysis and molecular signature of human retinal pigment epithelium.

Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed 'signature' genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases.

Receptor tyrosine kinase inhibitors AG013764 and AG013711 reduce choroidal neovascularization in rat eye.

Age-related macular degeneration (AMD) is the major cause of blindness for people over 60. In the "wet" form of AMD compounds targeting growth factor signaling pathways such as VEGF have been a major focus for therapeutic interventions. In a previously developed rat model of CNV, we utilized two receptor tyrosine kinase inhibitors (RTKi) to block VEGFR-1, VEGFR-2 and PDGFR signaling following the establishment of CNV. AAV-VEGF(165) was injected into the subretinal space of rats at postnatal days 15-17. Six weeks later, a suspension of RTK inhibitors, AG013764 or AG013711, was injected intraperitoneally (IP, twice daily) or intravitreally (every five days) over a two week period. FITC-dextran whole-mounts of RPE-choroid-sclera were prepared after the animals were sacrificed. CNV area was quantified using Neurolucida to measure the hyperfluorescence on FITC-dextran whole-mounts. Histology and immunohistochemistry were performed as described previously. VEGF expression in control and treated eyes was confirmed by immunohistochemistry and histological sections indicated recovery of retinal morphology and CNV reduction in treated eyes. In the animals IP injected with AG013764 or AG013711 the mean CNV level was reduced by 25 to 33% compared to control, but this effect did not achieve statistical significance. Intravitreal injections of AG013764 or AG013711 reduced the level of CNV by approximately 60% compared to control (p<0.005 or p<0.05, respectively). These data show that two RTK inhibitors, AG013764 or AG013711, delivered intravitreally, significantly reduce blood vessel proliferation in this AAV-VEGF(165) model of CNV.

Characterization of a novel glycine-rich protein from the cell wall of maize silk tissues.

The isolation, characterization and regulation of expression of a maize silk-specific gene is described. zmgrp5 (Zea mays glycine-rich protein 5) encodes a 187 amino acid glycine-rich protein that displays developmentally regulated silk-specific expression. Northern, Western, in situ mRNA hybridization and transient gene expression analyses indicate that zmgrp5 is expressed in silk hair and in cells of the vascular bundle and pollen tube transmitting tissue elements. The protein is secreted into the extracellular matrix and is localized in the cell wall fraction mainly through interactions mediated by covalent disulphide bridges. Taken together, these results suggest that the protein may play a role in maintaining silk structure during development. This is the first documented isolation of a stigma-specific gene from maize, an important agronomic member of the Poaceae family.

Preparation of soybean seed samples for FT-IR microspectroscopy.

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4 degrees C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.

Experimental Bin Drenching System for Testing Biocontrol Agents to Control Postharvest Decay of Apples.

A portable drencher capable of drenching a single bin of fruit was built to simulate the commercial application of chemicals to harvested apples in small orchard operations in the central and eastern United States. The drencher required as little as 125 liters of the treatment solution and permitted various bin travel speeds. Wounded apples were placed midway between the bottom and top of the bin, in the center, and near the four corners of the bin (20 fruit per location) and covered with enough unwounded apples to fill the bin. The bins were drenched with a suspension containing Penicillium expansum at 2 × 104 conidia per ml in 2000, 5 × 103 conidia per ml in 2001, and 3 × 103 conidia per ml in 2002 and 2003. In 2000 and 2003, the additional treatments included a combination of P. expansum with the yeast Metschnikowia pulcherrima at ~;1.2 × 107 CFU/ml, and in 2003 a combination with 2% sodium bicarbonate (SB) or a mixture of the yeast and SB. After 3 months of storage at ~;2°C, at all P. expansum conidial concentrations, more than 90% of wounded fruit developed decay on 'Golden Delicious', 'Delicious', and 'Rome' apples in the 2000-02 experiments. In 2003, 66 and 33.1% of the wounded fruit developed decay on 'Delicious' and 'Golden Delicious', respectively. The application of the antagonist reduced decay to 39 and 3.3% on 'Golden Delicious' in 2000 and 2003, respectively, and to 26% on 'Delicious' in 2003. The addition of SB reduced decay on both cultivars and, in combination with the yeast, was the most effective treatment on 'Golden Delicious'. This portable drencher can be very useful for evaluating different treatments applied to apples after harvest at the commercial level.

Effect of Prohexadione-Calcium Dose Level on Shoot Growth and Fire Blight in Young Apple Trees.

Prohexadione-calcium suppresses both shoot growth and fire blight in apple. In young apple orchards, there are conflicting requirements to control fire blight and allow sufficient tree growth for tree establishment. Application of prohexadione-calcium to various cultivars of orchard-grown apple trees ranging in age from newly planted to fifth-leaf trees indicated that fewer high-dose (125 or 250 mg ·liter-1) applications of prohexadione-calcium provided a better balance between fire blight control and growth in young orchards than multiple low-dose (30 or 63 mg·liter-1) applications. The response of early-season shoot growth to prohexadione-calcium treatment dose was linear. However, trees that received high doses of prohexadione-calcium tended to grow more in the latter part of the season, resulting in little or no difference in total seasonal growth between trees that received a few high or multiple low doses of prohexadione-calcium. Enhancement of fire blight resistance by prohexadione-calcium was correlated with shoot growth suppression at the time of inoculation, and the resistance response to prohexadione-calcium treatment dose was linear. Fire blight management strategies that use prohexadione-calcium in young apple orchards are discussed.

Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin.

White lupin (Lupinus albus) grown under P deficiency displays a suite of highly coordinated adaptive responses. Included among these is secretion of copious amounts of acid phosphatase (APase). Although numerous reports document that plants secrete APases in response to P deficiency, little is known of the biochemical and molecular events involved in this process. Here we characterize the secreted APase protein, cDNA, and gene from white lupin. The secreted APase enzyme is a glycoprotein with broad substrate specificity. It is synthesized as a preprotein with a deduced M(r) of 52,000 containing a 31-amino acid presequence. Analysis of the presequence predicts that the protein is targeted to outside the cell. The processed protein has a predicted M(r) of 49,000 but migrates as a protein with M(r) of 70,000 on sodium dodecyl sulfate gels. This is likely due to glycosylation. Enhanced expression is fairly specific to proteoid roots of P-stressed plants and involves enhanced synthesis of both enzyme protein and mRNA. Secreted APase appears to be encoded by a single gene containing seven exons interrupted by six introns. The 5'-upstream putative promoter of the white lupin-secreted APase contains a 50-base pair region having 72% identity to an Arabidopsis APase promoter that is responsive to P deficiency. The white lupin-secreted APase promoter and targeting sequence may be useful tools for genetically engineering important proteins from plant roots.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia.

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na(+) channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance (R(T)) are -5.9 mV and 829 Omega x cm(2), respectively. The apical membrane potential (V(A)) is -40.7 mV, and the mean ratio of apical to basolateral membrane resistance (R(A)/R(B)) is 2.8. Apical amiloride hyperpolarized V(A) by 19.7 mV and tripled R(A)/R(B). A cAMP-elevating cocktail depolarized V(A) by 17.6 mV, decreased R(A)/R(B) by 60%, increased short-circuit current by 6 microA/cm(2), decreased R(T) by 155 Omega x cm(2), and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na(+) currents [linear current-voltage (I-V) relation] and forskolin-stimulated Cl(-) currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2--4 microl x cm(-2) x h(-1)). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na(+) transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl(-) transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na(+)] and [Cl(-)].

Epinephrine-induced increases in [Ca2+](in) and KCl-coupled fluid absorption in bovine RPE.

PURPOSE: To define the ionic basis for the apical epinephrine-induced increase of fluid absorption (J(V)) across isolated bovine RPE-choroid. METHODS: Epinephrine-induced changes in RPE [Ca2+](in) levels were monitored with the ratioing dye fura-2. Transepithelial potential, resistance, and unidirectional fluxes of (36)Cl, (86)Rb (K substitute), and (22)Na were simultaneously determined in paired tissues from the same eye mounted in modified Ussing flux chambers. Radioisotopes (5-7 microCi) were added to the apical bath of one tissue and the basal bath of the other, and the appearance of label in the opposite bath was measured. RESULTS: Apical epinephrine (100 nM) transiently increased [Ca2+](in) by 153 +/- 78 nM. This increase was inhibited by the alpha(1)-adrenoreceptor antagonist prazosin (1 microM) and blocked by CPA(5 microM), an inhibitor of endoplasmic reticulum Ca2+-adenosine triphosphatases (ATPases). Apical epinephrine (100 nM) more than doubled the net Cl absorption rate, increased net K ((86)Rb) absorption by fivefold, and tripled net fluid absorption (J(V)), as predicted by isotonic coupling between ion and fluid transport. The epinephrine-induced increases in ion and fluid transport were completely inhibited by apical bumetanide (100 microM). CONCLUSIONS: Epinephrine increased fluid absorption across bovine RPE by activating apical membrane alpha(1)-adrenergic receptors, increasing [Ca2+](in), and stimulating bumetanide-sensitive Na,K,2Cl uptake at the apical membrane and KCl efflux at the basolateral membrane.

Adrenergic receptor activated ion transport in human fetal retinal pigment epithelium.

PURPOSE: To identify the apical and basolateral membrane mechanisms and intracellular signaling pathways in human fetal retinal pigment epithelium (HRPE) that mediate membrane voltage and resistance changes caused by apical membrane adrenergic receptor activation. METHODS: Intact sheets of RPE-choroid from human fetal eyes were mounted in a modified Ussing chamber. Ringer's solution composition changes on the retina-facing and choroid-facing sides of the tissue were separately controlled. Intracellular microelectrodes recorded the membrane voltage and resistance changes after the addition of pharmacologic agents to the apical or basal baths. RESULTS: Apical adrenergic agonists, isoproterenol and epinephrine (10(-8) M), depolarized the basolateral membrane, decreased total tissue resistance (R:(t)) and increased the ratio of apical-to-basolateral membrane resistance (R:(A)/R:(B)). Experiments using antagonists for alpha(1) and ss adrenergic receptors, prazosin and propranolol, respectively, indicated that both receptor types were present. The epinephrine responses were inhibited by apical bumetanide and basal 4,4'-diisothiocyanostilbene-2,2' disulfonic acid (DIDS). A cocktail of cyclic adenosine monophosphate (cAMP)-elevating agents produced basolateral membrane voltage and resistance changes very similar to the isoproterenol responses. The cAMP-induced electrical responses were strongly inhibited by basal 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). Ionomycin (to elevate intercellular Ca(2+), [Ca(2+)](i)) produced electrical responses similar to those caused by epinephrine. The Ca(2+) responses were unaffected by NPPB but were inhibited by 3 mM DIDS in the basal bath. CONCLUSIONS: The results provide evidence for two apical membrane adrenergic receptors, alpha(1) and ss, activated by epinephrine and isoproterenol, respectively. The membrane voltage and resistance changes produced by these two agonists mimic those produced by elevating [Ca(2+)](i) and [cAMP](i), suggesting that these ubiquitous signaling molecules activate separate basolateral membrane Cl channels inhibited by DIDS and NPPB, respectively. These two receptors, the apical membrane NaK2Cl cotransporters and the basolateral membrane Cl channels form a complex of proteins that help mediate fluid absorption across human RPE.

The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean.

A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12 days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells.

Assessing capacity.

Health care professionals often encounter patients who refuse a recommended treatment plan. Overriding a patient's right to autonomy has ethical, legal, and moral consequences. Incapacity is the determination by a physician that a patient lacks the ability to make informed decisions about his or her health care. The fundamentals needed for evaluating and documenting patients' capacity to make decisions regarding their personal medical care are provided.

Alendronate and estrogen effects in postmenopausal women with low bone mineral density. Alendronate/Estrogen Study Group.

The bisphosphonate alendronate and conjugated equine estrogens are both widely used for the treatment of postmenopausal osteoporosis. Acting by different mechanisms, these two agents decrease bone resorption and thereby increase or preserve bone mineral density (BMD). The comparative and combined effects of these medications have not been rigorously studied. This prospective, double blind, placebo-controlled, randomized clinical trial examined the effects of oral alendronate and conjugated estrogen, in combination and separately, on BMD, biochemical markers of bone turnover, safety, and tolerability in 425 hysterectomized postmenopausal women with low bone mass. In addition, bone biopsy with histomorphometry was performed in a subset of subjects. Treatment included placebo, alendronate (10 mg daily), conjugated equine estrogen (CEE; 0.625 mg daily), or alendronate (10 mg daily) plus CEE (0.625 mg daily) for 2 yr. All of the women received a supplement of 500 mg calcium daily. At 2 yr, placebo-treated patients showed a mean 0.6% loss in lumbar spine BMD, compared with mean increases in women receiving alendronate, CEE, and alendronate plus CEE of 6.0% (P < 0.001 vs. placebo), 6.0% (P < 0.001 vs. placebo), and 8.3% (P < 0.001 vs. placebo and CEE; P = 0.022 vs. alendronate), respectively. The corresponding changes in total proximal femur bone mineral density were +4.0%, +3.4%, +4.7%, and +0.3% for the alendronate, estrogen, alendronate plus estrogen, and placebo groups, respectively. Both alendronate and CEE significantly decreased biochemical markers of bone turnover, specifically urinary N-telopeptide of type I collagen and serum bone-specific alkaline phosphatase. The alendronate plus CEE combination produced slightly greater decreases in these markers than either treatment alone, but the mean absolute values remained within the normal premenopausal range. Alendronate, alone or in combination with CEE, was well tolerated. In the subset of patients who underwent bone biopsies, histomorphometry showed normal bone histology with the expected decrease in bone turnover, which was somewhat more pronounced in the combination group. Thus, alendronate and estrogen produced favorable effects on BMD. Combined use of alendronate and estrogen produced somewhat larger increases in BMD than either agent alone and was well tolerated.

Localization of peroxidase mRNAs in soybean seeds by in situ hybridization.

The soybean Ep gene encodes an anionic peroxidase enzyme that accumulates in large amounts in seed coat tissues. We have isolated a second peroxidase gene, Prx2, that is also highly expressed in developing seed coat tissues. Sequence analysis of Prx2 cDNA indicates that this transcript encodes a cationic peroxidase isozyme that is far removed from Ep in peroxidase phylogeny. To determine the expression patterns for these two peroxidases in developing seeds, the abundance and localization of the Ep and Prx2 transcripts were compared by in situ hybridization. Results show the expression of Ep begins in a small number of cells flanking the vascular bundle in the seed coat, spreads to encircle the seed, and then migrates to the hourglass cells as they develop. Expression of Prx2 occurs throughout development in all cell layers of the seed coat, and is also evident in the pericarp and embryo. Nonetheless, the Ep-encoded enzyme accounts for virtually all of the peroxidase activity detected in mature seed coats. The Prx2 enzyme is either insoluble in a catalytically inactive form, or is subject to degradation during seed maturation.

Hydrophobic protein synthesized in the pod endocarp adheres to the seed surface.

Soybean (Glycine max [L.] Merr.) hydrophobic protein (HPS) is an abundant seed constituent and a potentially hazardous allergen that causes asthma in persons allergic to soybean dust. By analyzing surface extracts of soybean seeds with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal microsequencing, we determined that large amounts of HPS are deposited on the seed surface. The quantity of HPS present varies among soybean cultivars and is more prevalent on dull-seeded phenotypes. We have also isolated cDNA clones encoding HPS and determined that the preprotein is translated with a membrane-spanning signal sequence and a short hydrophilic domain. Southern analysis indicated that multiple copies of the HPS gene are present in the soybean genome, and that the HPS gene structure is polymorphic among cultivars that differ in seed coat luster. The pattern of HPS gene expression, determined by in situ hybridization and RNA analysis, shows that HPS is synthesized in the endocarp of the inner ovary wall and is deposited on the seed surface during development. This study demonstrates that a seed dust allergen is associated with the seed luster phenotype in soybean and that compositional properties of the seed surface may be altered by manipulating gene expression in the ovary wall.

NADH-glutamate synthase in alfalfa root nodules. Genetic regulation and cellular expression.

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric beta-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

cAMP-dependent absorption of chloride across airway epithelium.

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell 85: 229-236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.