Expression of K+-Cl- cotransporters in the alpha-cells of rat endocrine pancreas

The expression of K+-Cl- cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in alpha-cells, but not pancreatic beta-cells nor delta-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both alpha-cell and beta-cells. Exposure of isolated alpha-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 microM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in beta-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of alpha-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on beta-cell volume. In conclusion, KCCs are expressed in pancreatic alpha-cells and beta-cells. However, they make a significant contribution to volume homeostasis only in alpha-cells.     

Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.     

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.     

Flow cytometric DNA analysis of corneal epithelium

We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.     

Alterations in the cell cycle characteristics of granulosa cells during the periovulatory period: evidence of ovarian and oviductal influences

Granulosa cells at different stages of differentiation were collected from ovarian follicles and oviducts during the periovulatory period, and their nuclear DNA content was monitored by flow cytometry to establish their cell cycle characteristics (G0 + G1, S, G2 + M). The proportion of cells in the three phases of the cell cycle varied in characteristics patterns depending upon the time they were collected, before or following ovulation. Granulosa (cumulus) cells recovered from ovulated oocytes were mitotically inactive as shown by the large proportion of cells with a 2C amount of DNA and the absence of cells in S phase. The proportion of granulosa cells in G2 + M decreased when recovery from the oviducts was delayed. In contrast, granulosa (cumulus and/or mural) cells recovered from preovulatory follicles prior to luteinizing hormone (LH) exposure contained a considerable population of cells undergoing DNA synthesis, and a decreased proportion of cells with a 2C DNA content. Our findings indicate that granulosa cells undergo dynamic and characteristics changes in all cell cycle phases during the periovulatory period, within follicular and oviductal environments. Intrafollicular events appear to play a major role in controlling DNA synthesis, proliferation, and related cell cycle events in the granulosa cells. Flow cytometric techniques provide objective and detailed information on the cell cycle characteristics of granulosa cell populations at different stages of differentiation. Elucidation of the mechanisms regulating cell cycle parameters of granulosa cells and their physiological significance thus seems feasible.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.     

Contractile and cytoskeletal proteins of smooth muscle cells in rat, rabbit, and human arteries

The aim of this study was to determine whether similar populations of smooth muscle cells, in relation to contractile and cytoskeletal proteins, are present in normal and diseased human coronary arteries and normal and injured rat and rabbit arteries. Rat aortae and rabbit carotid arteries were de-endothelialised and the resulting neointimal thickening examined at set time points 2-24 weeks later. Immunohistochemistry revealed that arteries had three distinct populations of cells in respect to alpha-smooth muscle actin, smooth muscle myosin heavy chain and vimentin (staining intensities '-', '+' or '++' for each protein), but only two populations in respect to desmin ('-' and '+'). The different populations of cells were found in the neointima at all times after injury, in human atherosclerotic plaque and in the media of diseased, injured and uninjured vessels, although in different proportions. It was concluded that arteries of the human, rat and rabbit have cells with a wide spectrum of contractile and cytoskeletal proteins. Expression of the different proteins did not reflect the state of the artery after injury or during the disease process, and was not associated with the expansion of a subset of cells within the artery wall.     

Flow cytometric analysis of intestinal intraepithelial lymphocytes in a model of immunodeficiency in Wistar rats

BACKGROUND: We have shown, in a rat model of immunodeficiency, permanent alterations in the thymus and in the gut-associated lymphoid tissues. We observed by immunohistochemistry an increase in the number of gamma/delta+ T cells in the gut lamina propria and in the number of CD8alpha/alpha+, CD25+, gamma/delta+ subpopulations of intestinal intraepithelial lymphocytes (iIEL). The aim of the present study was to analyze the isolated rat iIEL by flow cytometry. Materials and Methods Cells from mesenteric lymph nodes were examined in parallel with isolated iIEL. After staining with different antibodies, samples were run on a FACScan flow cytometer. Background staining was evaluated using isotype controls. Data analysis was performed using Lysys II software (Becton Dickinson) and WinMDI 2.3 software. RESULTS: 1) CD8alpha/beta populations do not express TCRgamma/delta, 2) CD8alpha/alpha+ populations express TCRgamma/delta, and its percentage is significantly increased in R21, 3) CD8alpha/beta and CD8alpha/alpha iIEL express TCRalpha/beta, being the percentage of CD8alpha/alpha+ TCRalpha/beta+ iIEL increased and the percentage of CD8alpha/beta+ TCRalpha/beta+ iIEL decreased in R21, and 4) CD8alpha/alpha as well as CD8alpha/beta iIEL do express CD25 only in R21. CONCLUSIONS: Considering the above results, we conclude that there exists an "in situ" origin and extrathymic maturation of the CD8alpha/alpha+ iIEL in the intestinal epithelium. The increase of TCRgamma/delta+ T cells may be triggered by the carbohydrate dextrin, to provide immune protection and control of inflammation at the intestinal level.     

Dopamine increases lung liquid clearance during mechanical ventilation

Short-term mechanical ventilation with high tidal volume (HVT) causes mild to moderate lung injury and impairs active Na+ transport and lung liquid clearance in rats. Dopamine (DA) enhances active Na+ transport in normal rat lungs by increasing Na+-K+-ATPase activity in the alveolar epithelium. We examined whether DA would increase alveolar fluid reabsorption in rats ventilated with HVT for 40 min compared with those ventilated with low tidal volume (LVT) and with nonventilated rats. Similar to previous reports, HVT ventilation decreased alveolar fluid reabsorption by ~50% (P < 0.001). DA increased alveolar fluid reabsorption in nonventilated control rats (by ~60%), LVT ventilated rats (by approximately 55%), and HVT ventilated rats (by ~200%). In parallel studies, DA increased Na+-K+-ATPase activity in cultured rat alveolar epithelial type II cells (ATII). Depolymerization of cellular microtubules by colchicine inhibited the effect of DA on HVT ventilated rats as well as on Na+-K+-ATPase activity in ATII cells. Neither DA nor colchicine affected the short-term Na+-K+-ATPase alpha1- and beta1-subunit mRNA steady-state levels or total alpha1- and beta1-subunit protein abundance in ATII cells. Thus we reason that DA improved alveolar fluid reabsorption in rats ventilated with HVT by upregulating the Na+-K+-ATPase function in alveolar epithelial cells.     

CD2 is expressed by a subpopulation of normal B cells and is frequently present in mature B-cell neoplasms

BACKGROUND: CD2 is expressed by T and natural killer (NK) cells and has been reported in T/NK cell lineage neoplasms as well as in immature B-lymphoblastic and myeloid leukemias. Although CD2+ B-cells have been identified in normal fetal and postnatal thymus, they have not been reported in adults. METHODS: We retrospectively reviewed flow cytometric immunophenotypic data on consecutive low-grade B-cell leukemias and lymphomas to investigate the frequency of CD2 expression. We also reviewed samples from normal healthy donors to determine whether there is a normal CD2+ B-cell population. RESULTS: CD2 expression (partial or complete) was observed in 13 of 83 (16%) chronic lymphocytic leukemias (CLL), 16 of 29 (55%) follicle center lymphomas (FCL), 3 of 12 (25%) hairy cell leukemias (HCL), 0 of 6 mantle cell lymphomas (MCL), 8 of 28 (29%) large cell lymphomas (LCL), and in 0 of 5 marginal zone/mucosa-associated lymphoid tissue lymphomas (MZL/MALT). We determined that 5.74 +/- 2.46% (mean +/- SD) of normal peripheral blood B cells and 6.48 +/- 1.62 % (mean +/- SD) of normal bone marrow B cells coexpress CD2. CONCLUSIONS: CD2 expression in B-cell neoplasia is a more prevalent phenomenon than previously appreciated. Normal CD2+ B-cell populations are observed in adults and may represent the nonmalignant counterpart of CD2+ B-cell neoplasms.     

Simultaneous detection of cyclin B1, p105, and DNA content provides complete cell cycle phase fraction analysis of cells that endoreduplicate

BACKGROUND: DNA analysis of endoreduplicating cells is difficult because of the overlap between stem-line G2 + M cells and 4C G1 cells. Simultaneous flow cytometry of DNA and cyclin B1 analytically separates these populations. The objective here was to develop simultaneous flow cytometry of DNA, cyclin B1, and p105 (highly expressed in mitosis) for improved, complete cell cycle phase fraction analysis of endoreduplicating cell populations. METHODS: Monoclonal antibody, GNS-1, reactive with human cyclin B1, was conjugated with fluorescein at three different fluorochrome-to-protein (F/P) ratios and tested for optimal sensitivity in a flow cytometric assay. A formaldehyde-methanol fixation procedure was optimized for retention of p105 within mitotic cells by analytic titration of formaldehyde. p105 was stained indirectly with Cy5-conjugated secondary antibody, followed by GNS-1, and DNA was stained with Hoechst 33342. The specificity of p105 in this assay was tested by comparison of manual and flow cytometric mitotic indices and by sorting and microscopic inspection. RESULTS: F/P 4.1 provided optimal fluorescein labeling of GNS-1. Formaldehyde (0.5%), followed by methanol permeabilization, fixed cells sufficiently to quantify stem-line and endoreduplicated G1, S, G2, and M phase fractions. Kinetic measurements of these fractions for both populations were demonstrated. CONCLUSIONS: The fluorochrome-to-protein ratio is important and can be optimized objectively for these assays. A permeabilization-sensitive antigen (p105), previously requiring formaldehyde/detergent-fixed cell preparations, was shown to work equally well with formaldehyde/ methanol fixation. Three-laser, two-parameter intracellular antigen analysis can be successfully coupled with DNA content analysis. Cell cycle kinetic analysis of endoreduplicating populations should be improved.     

Islet cell response in the neonatal rat after exposure to a high-fat diet during pregnancy

Although pancreatic beta-cells are capable of adapting their mass in response to insulin requirements, evidence has shown that a dietary insult could compromise this ability. Fetal malnutrition has been linked to low birth weight and the development of type 2 diabetes later in life, while reduced beta-cell mass has been reported in adult rats fed a high-fat diet (HFD). Reported here are the effects of exposure to a HFD, during different periods of gestation, on neonatal rat weight and beta- and alpha-cell development. The experimental groups were composed of neonatal offspring obtained from Wistar rats fed a high-fat (40% as energy) diet for either the first (HF1), second (HF2), or third (HF3) week, or all three (HF1-3) weeks of gestation. Neonatal weights and circulating glucose and insulin concentrations were measured on postnatal day 1, after which the pancreata were excised and processed for histological immunocytochemical examination and image analysis. HF1 and HF2 neonates were hypoglycemic, whereas HF1-3 neonates were hyperglycemic. Low birth weights were observed only in HF1 neonates. No significant differences were detected in the circulating insulin concentrations in the neonates, although beta-cell volume and numbers were reduced in HF1-3 neonates. beta-cell numbers also declined in HF1 and HF3 neonates. alpha-cell volume, number and size were, however, increased in HF1-3 neonates. alpha-cell size was also increased in HF1 and HF3 neonates. In neonates, exposure to a maternal HFD throughout gestation was found to have the most adverse effect on beta-cell development and resulted in hyperglycemia.     

Initial chromosome damage but not DNA damage is greater in ataxia telangiectasia cells.

Cells derived from individuals with ataxia telangiectasia (AT) exhibit increased sensitivity to ionizing radiation and certain drugs (e.g., bleomycin, neocarzinostatin, and etoposide) as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. To understand better the basis of this sensitivity, three AT and two normal lymphoblastoid cell lines were fractionated into cell cycle phase-enriched populations by centrifugal elutriation and then examined for their survival and their relative initial levels of DNA damage (neutral DNA filter elution) and chromosome damage (premature chromosome condensation). AT cells exhibited decreased levels of survival in all phases of the cell cycle; however, AT cells in early G1 phase were especially sensitive compared with normal cells in G1 phase. While AT and normal cells exhibited similar levels of initial DNA double-strand breaks in exponential populations as well as throughout the cell cycle, AT cells showed nearly twofold higher initial levels of chromosome damage than normal control cells in G1 and G2 phase. These results suggest that there is a higher rate of conversion of DNA double-strand breaks into chromosome breaks in AT cells, perhaps due to a difference in chromatin organization or stability. Thus one determining component of cellular radiosensitivity might include chromatin structure.     

Derivation of the frequency distributions of cycle durations from continuous labeling curves

In cell populations that are continuously exposed to radioactive thymidine over a long period, all proliferating cells become labeled as they pass through their DNA-replicating phase. The continuous labeling curve (CLC) shows the percentage of labeled cells versus time. Expected CLCs are calculated for cell populations with arbitrary frequency distributions of cycle durations. By optimizing the parameter values of a general probability function in the formula for CLC, frequency distributions of cycle durations are estimated from experimental CLCs. The analysis of several fetal rat tissues shows that the cycle durations vary over quite a wide range within the same tissue.     

Angiotensin II mediates beta endorphin like immunoactivity release in rat pituitary cell culture

The effect of angiotensin II (Ang II) on pituitary beta endorphin like immunoactivity (beta END-LI) release was studied in monolayer culture of normal rat pituicites. Ang II stimulated beta END-LI release into the culture media. This release of beta END-LI increased with longer incubation time and with higher doses of Ang II. The beta END-LI response was similar to the pattern of Ang II mediated ACTH release. Ang II stimulated beta END-LI release was blocked by cycloheximide and decreased by corticosterone (5 nmol/l). Successively higher concentrations of [SAR GLY]Ang II, a known Ang II antagonist, induced greater inhibition of Ang II stimulated beta END-LI release. Gel chromatography of pooled media from control and Ang II stimulated cells revealed three peaks of beta END-LI which migrated with the void volume, beta lipotropin (beta LPH) and beta endorphin. The relative amount of beta END-LI in these peaks [(BEND-LI peak + total beta END-LI in column) x 100%] from media of control and stimulated cells were as follows: (1) Void 7% and 19% (2) beta LPH 50% and 52% (3) beta endorphin 43% and 29%.     

Phenotypic characterization of murine tumor-infiltrating T lymphocytes.

Tumor infiltrating lymphocytes (TIL) can be isolated from solid tumors and selectively expanded in long term culture with IL-2 and autologous irradiated tumor. Such long term cultured cells express anti-tumor activity in vitro, mediate the regression of established tumor in murine models of cancer, and have been used for the treatment of cancer in humans. We have characterized freshly isolated mouse Thy-1+ TIL populations, as well as long term TIL cultures, from several different C57BL/6 (B6) tumors. Freshly isolated Thy-1+ TIL include both CD4+ and CD8+ cells, as well as cells bearing NK markers. These cells are predominantly TCR alpha beta+, with a smaller population of TCR gamma delta+ cells. The TCR alpha beta+ cells expressed a broad distribution of V beta phenotypes that was statistically different from that expressed in normal B6 splenic Thy-1+ cells or CD8+ cells, presumably reflecting in vivo selection in the host anti-tumor response. NK cells are present in these tumors at a greater frequency than noted in splenic T cells. Cultured TIL populations rapidly became exclusively Thy-1+/CD8+/CD4- and TCR alpha beta+/gamma delta-. Individual long term TIL populations initially expressed multiple V beta products, but rapidly restricted their V beta expression, frequently expressing a single dominant V beta. The identity of this dominant V beta varied among different TIL lines, but the overall representation of V beta phenotypes in these cultures was statistically different from that seen in Thy-1+ or CD8+ splenocytes. No statistical difference was noted between lines derived from antigenically distinct tumors. The selection of tumor specific T cells in vitro is therefore not reflected in any simple predominance of V beta usage. The complexity of TCR usage in the anti-tumor response may result from the involvement of multiple alpha- and beta-chain regions in the response to a single antigenic determinant, or may reflect multiple antigenic determinants expressed on a single syngeneic tumor.     

Gradient fractionation of cycling and resting cells monitored by BrdUrd incorporation

A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium Iodide (PI), showed the S compartment highly concentrated in the 1.073/77 g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations.     

beta]-Tubulin Accumulation and DNA Replication in Imbibing Tomato Seeds

The activation of the cell cycle in embryo root tips of imbibing tomato (Lycopersicon esculentum Mill. cv Lerica) seeds was studied by flow cytometric analyses of the nuclear DNA content and by immunodelection of [beta]-tubulin. With dry seeds, flow cytometric profiles indicated that the majority of the cells were arrested at the G1 phase of the cell cycle. In addition, [beta]-tubulin was not detectable on western blots. Upon imbibition of water, the number of cells in G2 started to increase after 24 h, and a 55-kD [beta]-tubulin signal was detected between 24 and 48 h. Two-dimensional immunoblots revealed at least three different [beta]-tubulin isotypes. Thus, [beta]-tubulin accumulation and DNA replication were induced during osmotic priming. These processes, as well as seed germination rate, were enhanced upon subsequent imbibition of water, compared with control seeds that imbibed but were not primed. By contrast, when aged seeds imbibed, DNA replication, [beta]-tubulin accumulation, and germination were delayed. In all cases studied, both DNA replication and [beta]-tubulin accumulation preceded visible germination. We suggest that activation of these cell-cycle-related processes is a prerequisite for tomato seed germination. Furthermore, [beta]-tubulin expression can be used as a parameter for following the initial processes that are activated during seed imbibition.     

Unusual T cell populations in adult murine bone marrow. Prevalence of CD3+CD4-CD8- and alpha beta TCR+NK1.1+ cells

The T cell populations present in normal murine bone marrow have not been previously analyzed in detail, mainly because of their relative rarity. In order to permit such analyses, bone marrow T cells were enriched by depleting Mac1-positive cells, which constitute 65 to 90% of bone marrow cells (BMC), and then studied by two-color flow cytometry. Analysis of the remaining cells revealed that the T cell profile of adult murine bone marrow is markedly different from that of other lymphoid organs. A very high proportion of bone marrow CD3+ cells (approximately one-third) are CD4-CD8-. CD3+CD4-CD8- cells are much more concentrated among BMC T cells than among thymocytes or splenic T cells, suggesting that bone marrow may be either a site of extrathymic TCR gene rearrangement, or a major site to which such cells home from the thymus. The expression of NK1.1 was also evaluated on Mac1-depleted BMC populations. Surprisingly, up to 39% of alpha beta TCR+ BMC were found to express NK1.1. Most alpha beta TCR+NK1.1+ BMC also expressed CD4 or CD8. NK1.1+ alpha beta TCR+ cells represented a much greater proportion of BMC T cells than of other lymphoid (splenocyte or thymocyte) T cell populations. Mac1-depleted BMC of nude mice contained very few cells with this phenotype. These results are consistent with the hypothesis that NK1.1+ alpha beta TCR+ cells are generated primarily in the thymus of normal animals and migrate preferentially to bone marrow, where they may function as regulatory elements in hematopoiesis.