Genes Involved in Osmoregulation during Turgor-Driven Cell Expansion of Developing Cotton Fibers Are Differentially Regulated1

Cotton (Gossypium hirsutum L.) fibers are single-celled trichomes that synchronously undergo a phase of rapid cell expansion, then a phase including secondary cell wall deposition, and finally maturation. To determine if there is coordinated regulation of gene expression during fiber expansion, we analyzed the expression of components involved in turgor regulation and a cytoskeletal protein by measuring levels of mRNA and protein accumulation and enzyme activity. Fragments of the genes for the plasma membrane proton-translocating ATPase, vacuole-ATPase, proton-translocating pyrophosphatase (PPase), phosphoenolpyruvate carboxylase, major intrinsic protein, and α-tubulin were amplified by polymerase chain reaction and used as probes in ribonuclease protection assays of RNA from a fiber developmental series, revealing two discrete patterns of mRNA accumulation. Transcripts of all but the PPase accumulated to highest levels during the period of peak expansion (+12–15 d postanthesis [dpa]), then declined with the onset of secondary cell wall synthesis. The PPase was constitutively expressed through fiber development. Activity of the two proton-translocating-ATPases peaked at +15 dpa, whereas PPase activity peaked at +20 dpa, suggesting that all are involved in the process of cell expansion but with varying roles. Patterns of protein accumulation and enzyme activity for some of the proteins examined suggest posttranslational regulation through fiber development.

Spatial and Temporal Analyses of Expansion and Cell Cycle in Sunflower Leaves1 : A Common Pattern of Development for All Zones of a Leaf and Different Leaves of a Plant

We have investigated the spatial distributions of expansion and cell cycle in sunflower (Helianthus annuus L.) leaves located at two positions on the stem, from leaf initiation to the end of expansion. Relative expansion rate (RER) was analyzed by following the deformation of a grid drawn on the lamina; relative division rate (RDR) and flow-cytometry data were obtained in four zones perpendicular to the midrib. Calculations for determining in situ durations of the cell cycle and of S-G2-M in the epidermis are proposed. Area and cell number of a given leaf zone increased exponentially during the first two-thirds of the development duration. RER and RDR were constant and similar in all zones of a leaf and in all studied leaves during this period. Reduction in RER occurred afterward with a tip-to-base gradient and lagged behind that of RDR by 4 to 5 d in all zones. After a long period of constancy, cell-cycle duration increased rapidly and simultaneously within a leaf zone, with cells blocked in the G0-G1 phase of the cycle. Cells that began their cycle after the end of the period with exponential increase in cell number could not finish it, suggesting that they abruptly lost their competence to cross a critical step of the cycle. Differences in area and in cell number among zones of a leaf and among leaves of a plant essentially depended on the timing of two events, cessation of exponential expansion and of exponential division.

Repeat expansion by homologous recombination in the mouse germ line at palindromic sequences

Genetic instability can be induced by unusual DNA structures and sequence repeats. We have previously demonstrated that a large palindrome in the mouse germ line derived from transgene integration is extremely unstable and undergoes stabilizing rearrangements at high frequency, often through deletions that produce asymmetry. We have now characterized other palindrome rearrangements that arise from complex homologous recombination events. The structure of the recombinants is consistent with homologous recombination occurring by a noncrossover gene conversion mechanism in which a break induced in the palindrome promotes homologous strand invasion and repair synthesis, similar to mitotic break repair events reported in mammalian cells. Some of the homologous recombination events led to expansion in the size of the palindromic locus, which in the extreme case more than doubled the number of repeats. These results may have implications for instability observed at naturally occurring palindromic or quasipalindromic sequences.

Expansion of a recurrent V beta 5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients.

We have used a PCR-based technology to study the V beta 5 and V beta 17 repertoire of T-cell populations in HLA-DR2 multiple sclerosis (MS) patients. We have found that the five MS DR2 patients studied present, at the moment of diagnosis and prior to any treatment, a marked expansion of a CD4+ T-cell population bearing V beta 5-J beta 1.4 beta chains. The sequences of the complementarity-determining region 3 of the expanded T cells are highly homologous. One shares structural features with that of the T cells infiltrating the central nervous system and of myelin basic protein-reactive T cells found in HLA-DR2 MS patients. An homologous sequence was not detectable in MS patients expressing DR alleles other than DR2. However, it is detectable but not expanded in healthy DR2 individuals. The possible mechanisms leading to its in vivo proliferation at the onset of MS are discussed.

The heart communicates with the kidney exclusively through the guanylyl cyclase-A receptor: acute handling of sodium and water in response to volume expansion.

Disruption of guanylyl cyclase-A (GC-A) results in mice displaying an elevated blood pressure, which is not altered by high or low dietary salt. However, atrial natriuretic peptide (ANP), a proposed ligand for GC-A, has been suggested as critical for the maintenance of normal blood pressure during high salt intake. In this report, we show that infusion of ANP results in substantial natriuresis and diuresis in wild-type mice but fails to cause significant changes in sodium excretion or urine output in GC-A-deficient mice. ANP, therefore, appears to signal through GC-A in the kidney. Other natriuretic/diuretic factors could be released from the heart. Therefore, acute volume expansion was used as a means to cause release of granules from the atrium of the heart. That granule release occurred was confirmed by measurements of plasma ANP concentrations, which were markedly elevated in both wild-type and GC-A-null mice. After volume expansion, urine output as well as urinary sodium and cyclic GMP excretion increased rapidly and markedly in wild-type mice, but the rapid increases were abolished in GC-A-deficient animals. These results strongly suggest that natriuretic/diuretic factors released from the heart function exclusively through GC-A.

Activated Ras signals differentiation and expansion of CD4+8+ thymocytes.

We describe a novel approach to assay the ability of particular gene products to signal transitions in lymphocyte differentiation in vivo. The method involves transfection of test expression constructs into RAG-1-deficient embryonic stem cells, which are subsequently assayed by the RAG-2-deficient blastocyst complementation approach. We have used this method to demonstrate that expression of activated Ras in CD4-8- (double negative, DN) prothymocytes in vivo induces their differentiation into small CD4+8+ (double positive, DP) cortical thymocytes with accompanying expansion to normal thymocyte numbers. However, activated Ras expression in DP cells does not cause proliferation or maturation to CD4+8- or CD4-8+ (single positive) thymocytes. Therefore, signaling through Ras is sufficient for promoting differentiation of DN to DP cells, but further differentiation requires the activity of additional signaling pathways.

Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin.

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.

Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy.

The conventional approach to cytotoxic T-lymphocyte (CTL) induction uses maximal antigen concentration with the intent of eliciting more CTL. However, the efficacy of this approach has not been systematically explored with regard to the quality of the CTLs elicited or their in vivo functionality. Here, we show that a diametrically opposite approach elicits CTLs that are much more effective at clearing virus. CTLs specific for a defined peptide epitope were selectively expanded with various concentrations of peptide antigen. CTLs generated with exceedingly low-dose peptide lysed targets sensitized with > 100-fold less peptide than CTLs generated with high-dose peptide. Differences in expression of T-cell antigen receptors or a number of other accessory molecules did not account for the functional differences. Further, high-avidity CTLs adoptively transferred into severe combined immunodeficient mice were 100- to 1000-fold more effective at viral clearance than the low-avidity CTLs, despite the fact that all CTL lines lysed virus-infected targets in vitro. Thus, the quality of CTLs is as important as the quantity of CTLs for adoptive immunotherapy, and the ability to kill virally infected targets in vitro is not predictive of in vivo efficacy, whereas the determinant density requirement described here is predictive. Application of these principles may be critical in developing effective adoptive cellular immunotherapy for viral infections and cancer.

Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.

Oxytocin mediates atrial natriuretic peptide release and natriuresis after volume expansion in the rat.

Our previous studies have shown that stimulation of the anterior ventral third ventricular region increases atrial natriuretic peptide (ANP) release, whereas lesions of this structure, the median eminence, or removal of the neural lobe of the pituitary block ANP release induced by blood volume expansion (BVE). These results indicate that participation of the central nervous system is crucial in these responses, possibly through mediation by neurohypophysial hormones. In the present research we investigated the possible role of oxytocin, one of the two principal neurohypophysial hormones, in the mediation of ANP release. Oxytocin (1-10 nmol) injected i.p. caused significant, dose-dependent increases in urinary osmolality, natriuresis, and kaliuresis. A delayed antidiuretic effect was also observed. Plasma ANP concentrations increased nearly 4-fold (P < 0.01) 20 min after i.p. oxytocin (10 nmol), but there was no change in plasma ANP values in control rats. When oxytocin (1 or 10 nmol) was injected i.v., it also induced a dose-related increase in plasma ANP at 5 min (P < 0.001). BVE by intra-atrial injection of isotonic saline induced a rapid (5 min postinjection) increase in plasma oxytocin and ANP concentrations and a concomitant decrease in plasma arginine vasopressin concentration. Results were similar with hypertonic volume expansion, except that this induced a transient (5 min) increase in plasma arginine vasopressin. The findings are consistent with the hypothesis that baroreceptor activation of the central nervous system by BVE stimulates the release of oxytocin from the neurohypophysis. This oxytocin then circulates to the right atrium to induce release of ANP, which circulates to the kidney and induces natriuresis and diuresis, which restore body fluid volume to normal levels.

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure.

Myotonic dystrophy is caused by an expansion of a CTG triplet repeat sequence in the 3' noncoding region of a protein kinase gene, yet the mechanism by which the triplet repeat expansion causes disease remains unknown. This report demonstrates that a DNase I hypersensitive site is positioned 3' of the triplet repeat in the wild-type allele in both fibroblasts and skeletal muscle cells. In three unrelated individuals with myotonic dystrophy that have large expansions of the triplet repeat, the allele with the triplet repeat expansion exhibited both overall DNase I resistance and inaccessibility of nucleases to the adjacent hypersensitive site. These results indicate that the triplet repeat expansion alters the adjacent chromatin structure, establishing a region of condensed chromatin, and suggests a molecular mechanism for myotonic dystrophy.

Soil quality response to land-use change for sugarcane expansion in Brazil

Globally, increasing demands for biofuels have intensified the rate of land-use change (LUC) for expansion of bioenergy crops. In Brazil, the world\'s largest sugarcane-ethanol producer, sugarcane area has expanded by 35% (3.2 Mha) in the last decade. Sugarcane expansion has resulted in extensive pastures being subjected to intensive mechanization and large inputs of agrochemicals, which have direct implications on soil quality (SQ). We hypothesized that LUC to support sugarcane expansion leads to overall SQ degradation. To test this hypothesis we conducted a field-study at three sites in the central-southern region, to assess the SQ response to the primary LUC sequence (i.e., native vegetation to pasture to sugarcane) associated to sugarcane expansion in Brazil. At each land use site undisturbed and disturbed soil samples were collected from the 0-10, 10-20 and 20-30 cm depths. Soil chemical and physical attributes were measured through on-farm and laboratory analyses. A dataset of soil biological attributes was also included in this study. Initially, the LUC effects on each individual soil indicator were quantified. Afterward, the LUC effects on overall SQ were assessed using the Soil Management Assessment Framework (SMAF). Furthermore, six SQ indexes (SQI) were developed using approaches with increasing complexity. Our results showed that long-term conversion from native vegetation to extensive pasture led to soil acidification, significant depletion of soil organic carbon (SOC) and macronutrients [especially phosphorus (P)] and severe soil compaction, which creates an unbalanced ratio between water- and air-filled pore space within the soil and increases mechanical resistance to root growth. Conversion from pasture to sugarcane improved soil chemical quality by correcting for acidity and increasing macronutrient levels. Despite those improvements, most of the P added by fertilizer accumulated in less plant-available P forms, confirming the key role of organic P has in providing available P to plants in Brazilian soils. Long-term sugarcane production subsequently led to further SOC depletions. Sugarcane production had slight negative impacts on soil physical attributes compared to pasture land. Although tillage performed for sugarcane planting and replanting alleviates soil compaction, our data suggested that the effects are short-term with persistent, reoccurring soil consolidation that increases erosion risk over time. These soil physical changes, induced by LUC, were detected by quantitative soil physical properties as well as by visual evaluation of soil structure (VESS), an on-farm and user-friendly method for evaluating SQ. The SMAF efficiently detected overall SQ response to LUC and it could be reliably used under Brazilian soil conditions. Furthermore, since all of the SQI values developed in this study were able to rank SQ among land uses. We recommend that simpler and more cost-effective SQI strategies using a small number of carefully chosen soil indicators, such as: pH, P, K, VESS and SOC, and proportional weighting within of each soil sectors (chemical, physical and biological) be used as a protocol for SQ assessments in Brazilian sugarcane areas. The SMAF and SQI scores suggested that long-term conversion from native vegetation to extensive pasture depleted overall SQ, driven by decreases in chemical, physical and biological indicators. In contrast, conversion from pasture to sugarcane had no negative impacts on overall SQ, mainly because chemical improvements offset negative impacts on biological and physical indicators. Therefore, our findings can be used as scientific base by farmers, extension agents and public policy makers to adopt and develop management strategies that sustain and/or improving SQ and the sustainability of sugarcane production in Brazil.