Root Xylem Embolisms and Refilling. Relation to Water Potentials of Soil, Roots, and Leaves, and Osmotic Potentials of Root Xylem Sap1

Embolism and refilling of vessels was monitored directly by cryomicroscopy of field-grown corn (Zea mays L.) roots. To test the reliability of an earlier study showing embolism refilling in roots at negative leaf water potentials, embolisms were counted, and root water potentials (Ψroot) and osmotic potentials of exuded xylem sap from the same roots were measured by isopiestic psychrometry. All vessels were full at dawn (Ψroot −0.1 MPa). Embolisms were first seen in late metaxylem vessels at 8 am. Embolized late metaxylem vessels peaked at 50% at 10 am (Ψroot −0.1 MPa), fell to 44% by 12 pm (Ψroot −0.23 MPa), then dropped steadily to zero by early evening (Ψroot −0.28 MPa). Transpiration was highest (8.5 μg cm−2 s−1) between 12 and 2 pm when the percentage of vessels embolized was falling. Embolized vessels were refilled by liquid moving through their lateral walls. Xylem sap was very low in solutes. The mechanism of vessel refilling, when Ψroot is negative, requires further investigation. Daily embolism and refilling in roots of well-watered plants is a normal occurrence and may be a component of an important hydraulic signaling mechanism between roots and shoots.

Potential responses of soil organic carbon to global environmental change

Recent improvements in our understanding of the dynamics of soil carbon have shown that 20–40% of the approximately 1,500 Pg of C stored as organic matter in the upper meter of soils has turnover times of centuries or less. This fast-cycling organic matter is largely comprised of undecomposed plant material and hydrolyzable components associated with mineral surfaces. Turnover times of fast-cycling carbon vary with climate and vegetation, and range from <20 years at low latitudes to >60 years at high latitudes. The amount and turnover time of C in passive soil carbon pools (organic matter strongly stabilized on mineral surfaces with turnover times of millennia and longer) depend on factors like soil maturity and mineralogy, which, in turn, reflect long-term climate conditions. Transient sources or sinks in terrestrial carbon pools result from the time lag between photosynthetic uptake of CO2 by plants and the subsequent return of C to the atmosphere through plant, heterotrophic, and microbial respiration. Differential responses of primary production and respiration to climate change or ecosystem fertilization have the potential to cause significant interrannual to decadal imbalances in terrestrial C storage and release. Rates of carbon storage and release in recently disturbed ecosystems can be much larger than rates in more mature ecosystems. Changes in disturbance frequency and regime resulting from future climate change may be more important than equilibrium responses in determining the carbon balance of terrestrial ecosystems.

Correction of hypertension by normalization of endothelial levels of fibroblast growth factor and nitric oxide synthase in spontaneously hypertensive rats.

Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. Restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

Correction of diabetic alterations by glucokinase.

Hyperglycemia is a common feature of diabetes mellitus. It results from a decrease in glucose utilization by the liver and peripheral tissues and an increase in hepatic glucose production. Glucose phosphorylation by glucokinase is an initial event in glucose metabolism by the liver. However, glucokinase gene expression is very low in diabetic animals. Transgenic mice expressing the P-enolpyruvate carboxykinase/glucokinase chimeric gene were generated to study whether the return of the expression of glucokinase in the liver of diabetic mice might prevent metabolic alterations. In contrast to nontransgenic mice treated with streptozotocin, mice with the transgene previously treated with streptozotocin showed high levels of both glucokinase mRNA and its enzyme activity in the liver, which were associated with an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in gluconeogenesis and ketogenesis in the liver and the production of glucose and ketone body by hepatocytes in primary culture were observed in streptozotocin-treated transgenic mice. Thus, glycolysis was induced while gluconeogenesis and ketogenesis were blocked in the liver of diabetic mice expressing glucokinase. This was associated with normalization of blood glucose, ketone bodies, triglycerides, and free fatty acids even in the absence of insulin. These results suggest that the expression of glucokinase during diabetes might be a new approach to the normalization of hyperglycemia.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.

Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene.

The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to cisplatin and gamma-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is approximately 2.5 kb and detects an approximately 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels.

Estado nutricional de cultivares de cana-de-açúcar em função da aplicação de calcário e gesso no solo

As áreas destinadas à cultura de cana-de-açúcar estão concentradas em áreas de Cerrado, que são solos ácidos, com elevada saturação por alumínio e baixa saturação por bases. Para correção destas características, faz-se o uso de corretivos e fertilizantes no momento da implantação da cultura. Embora a toxidez por alumínio seja uma das mais sérias limitações ao crescimento de plantas em solos ácidos, há uma carência de estudos relacionando-a com a cultura de cana-de-açúcar. Com base nisto, o objeto deste trabalho foi avaliar o efeito da aplicação de calcário e gesso sobre o estado nutricional de quatro cultivares de cana-de-açúcar, distintas quanto à exigência nutricional, na fase de perfilhamento da cultura. Para tanto, desenvolveu-se experimento em casa de vegetação com vasos contendo 4 kg de amostra de solo, com saturação por alumínio de 29%, como substrato, com os seguintes tratamentos: 1) aplicação de calcário, 2) aplicação de gesso, e 3) controle, sem adição de calcário e gesso. Os cultivares utilizados foram: IACSP95-5000, IACSP94-2094, SP80-3280 e IAC95-3028. Após adição dos tratamentos, as amostras de solo foram incubadas por 45 dias. Para os materiais vegetais, mini-toletes de gema única das cultivares foram pré-brotados, crescidos por 90 dias e transplantados para os vasos, para os respectivos tratamentos. As plantas foram adubadas com macro e micronutrientes e cultivadas por 60 dias. Os tratamentos foram caracterizados quanto aos atributos químicos para avaliação da fertilidade após a incubação e após a coleta das plantas. Nas plantas, foram feitas avaliações de: clorofila, flavonoides, índice de balanço do nitrogênio (NBI), concentração de alumínio e dos nutrientes e produção de matéria seca de folha, colmo e raiz. Enquanto o tratamento gesso não diferiu do controle, o tratamento calcário foi efetivo para correção da reação da amostra de solo, com elevação dos valores de pH e saturação por bases, e redução da saturação por alumínio e acidez total, além do aumento nos teores de Ca e Mg. A adubação NPK+micro foi efetiva para aumentar os teores de P e K dos substratos de todos os tratamentos. Nas plantas, para todos os cultivares, os índices de clorofila, flavonoides e NBI foram mais adequados no tratamento calcário, em relação ao controle e ao gesso. A produção de matéria seca total e das partes das plantas, de modo geral, foi maior para a IACSP95-5000, intermediaria para a IACSP35-3028 e menor para a IACSP94-2094 e SP80-3280. Quanto aos tratamentos, não houve efeito do calcário e gesso sobre a produção de matéria seca de folha, contudo, houve aumento de produção de matéria seca de colmo e raiz pelo calcário, na cultivar IACSP95-5000. Independente do tratamento, as cultivares apresentaram concentração de alumínio superior a 5000 mg kg-1 nas raízes e inferiores a 360 e 160 mg kg-1 nas folhas e colmos, respectivamente, evidenciando que o Al foi acúmulo nas raízes das plantas. De modo geral, as concentrações de macro e micronutrientes variaram com os tratamentos e cultivares, porém sem valores altos e baixos extremos. Concluiu-se que a cultivar IACSP95-5000 tem maior potencial de produção em resposta à aplicação de calcário e que a saturação de alumínio de 29% não apresenta efeito negativo sobre a produção de matéria seca, exceto da cultivar IACSP95-5000, e estado nutricional das cultivares de cana-de-açúcar

Feasibility of Soil Vapor Extraction Systems in Low Permeability Soils

Soil vapor extraction (SVE)systems can be used to remediate enviornmental sites that have been contaminated with petroleum products. However, SVE systems rely on pore space in soils to draw the vapors through the soil, creating a vacuum. Therefore, SVE systems are not as effective when used in low permeability soils. This study aims to determine whether SVE systems can be used on low permeability soils in conjunction with companion technologies. The results indicate that SVE systems can be utilized in low permeability soils if used in conjunction with companion technologies that increase soil permeability and cantaminant volatilization. The promising companion technology is six-phase soil heating, based on contamination removal rate and cost estimates.

Plant regeneration functional groups modulate the response to fire of soil enzyme activities in a Mediterranean shrubland

Soil enzymes are critical to soil nutrient cycling function but knowledge on the factors that control their response to major disturbances such as wildfires remains very limited. We evaluated the effect of fire-related plant functional traits (resprouting and seeding) on the resistance and resilience to fire of two soil enzyme activities involved in phosphorus and carbon cycling (acid phosphatase and β-glucosidase) in a Mediterranean shrublands in SE Spain. Using experimental fires, we compared four types of shrubland microsites: SS (vegetation patches dominated by seeder species), RR (patches dominated by resprouter species), SR (patches co-dominated by seeder and resprouter species), and IP (shrub interpatches). We assessed pre- and post-fire activities of the target soil enzymes, available P, soil organic C, and plant cover dynamics over three years after the fire. Post-fire regeneration functional groups (resprouter, seeder) modulated both pre- and post-fire activity of acid phosphatase and β-glucosidase, with higher activity in RR and SR patches than in SS patches and IP. However, we found no major differences in enzyme resistance and resilience between microsite types, except for a trend towards less resilience in SS patches. Fire similarly reduced the activity of both enzymes. However, acid phosphatase and β-glucosidase showed contrasting post-fire dynamics. While β-glucosidase proved to be rather resilient to fire, fully recovering three years after fire, acid phosphatase showed no signs of recovery in that period. Overall, the results indicate a positive influence of resprouter species on soil enzyme activity that is very resistant to fire. Long-lasting decrease in acid phosphatase activity probably resulted from the combined effect of P availability and post-fire drought. Our results provide insights on how plant functional traits modulate soil biochemical and microbiological response to fire in Mediterranean fire-prone shrublands.

New Approach for Correction of Error Associated With Keratometric Estimation of Corneal Power in Keratoconus

The aim of this study was to obtain the exact value of the keratometric index (nkexact) and to clinically validate a variable keratometric index (nkadj) that minimizes this error. Methods: The nkexact value was determined by obtaining differences (DPc) between keratometric corneal power (Pk) and Gaussian corneal power (PGauss c ) equal to 0. The nkexact was defined as the value associated with an equivalent difference in the magnitude of DPc for extreme values of posterior corneal radius (r2c) for each anterior corneal radius value (r1c). This nkadj was considered for the calculation of the adjusted corneal power (Pkadj). Values of r1c ∈ (4.2, 8.5) mm and r2c ∈ (3.1, 8.2) mm were considered. Differences of True Net Power with PGauss c , Pkadj, and Pk(1.3375) were calculated in a clinical sample of 44 eyes with keratoconus. Results: nkexact ranged from 1.3153 to 1.3396 and nkadj from 1.3190 to 1.3339 depending on the eye model analyzed. All the nkadj values adjusted perfectly to 8 linear algorithms. Differences between Pkadj and PGauss c did not exceed 60.7 D (Diopter). Clinically, nk = 1.3375 was not valid in any case. Pkadj and True Net Power and Pk(1.3375) and Pkadj were statistically different (P , 0.01), whereas no differences were found between PGauss c and Pkadj (P . 0.01). Conclusions: The use of a single value of nk for the calculation of the total corneal power in keratoconus has been shown to be imprecise, leading to inaccuracies in the detection and classification of this corneal condition. Furthermore, our study shows the relevance of corneal thickness in corneal power calculations in keratoconus.