The effect of slope on repeated sprint ability in young soccer players.

This study aimed to describe a gradient repeated sprint ability (RSA) test in comparison with a standard level one by investigating performance, metabolic demand and muscular jumping performance as a proxy for running mechanics. Eighteen athletes performed two level RSA tests (40 m × 6) - for reliability evaluation - and one ±5% gradient RSA test, second leg downhill (RSAgrad). Rating of perceived exertion (RPE), blood lactate concentration (BLa) concentration, vertical jump heights were assessed as well. Level test measures resulted highly reliable (Intra-class correlation coefficient (ICC) ≥0.96). RSAgrad worsened only first sprints' performance (-2%) but not overall test performance (~45 s). RSAgrad resulted to be less deteriorating in terms of fatigue index (FI) (-36%), BLa (-23%), RPE (-11%), jumping performance (RSAgrad post-/pre-squat jump, countermovement jump heights (CMJh): -3%, -6%, respectively). RSAgrad could be used to diversify common training protocol without stressing excessively athletes' current metabolic-anaerobic capacity. Such physical conditioning procedures could improve acceleration/braking capability.

The acute effect of whole body vibration on repeated shuttle-running in young soccer players.

The aim of this study was to investigate the acute effects of whole-body vibration (WBV) on Repeated Sprint Ability (RSA). Seventeen male soccer players (16.71±0.47 y) performed three RSA tests (Randomized crossover study design). The second RSA test was done with WBV (RSA2) to assess the effect of WBV. The studied variables were: best time (BT), worst time (WT), total time (TT), the fatigue index (FI) of RSA, and post-test blood lactate (BLa). ANOVA with repeated measures showed no differences between RSA1 and RSA3, while there were significant differences in all variables studied. TT= [RSA2 0.93% and 1.68% lower than RSA1 and RSA3 respectively; p<0.05], BLa= [RSA2 16.97% and 14.73% greater than RSA1 and RSA3 respectively; p<0.001], WT= [RSA2 1.90% and 2.93% lower than RSA1 and RSA3 respectively; p<0.01], and FI = [RSA2 30.64% and 40.15% lower than RSA1 and RSA3 respectively; p<0.0001]. When comparing individual sprints, WBV showed a significant effect at the 5th sprint: RSA2 2.29% and 2.95% lower than RSA1 and RSA3 respectively (p<0.005), while at the 6th sprint: RSA2 2.75% and 4.09% lower than RSA1 and RSA3 respectively; p<0.005. In conclusion, when applying WBV during the recovery periods of Repeated Sprint Ability efforts, most of the performance variables improved.

The effects of one-week training camp on motor skills in Karate kids.

AIM: The purpose of this study was to evaluate the effects of short-term increased hours of specific high-intensity karate training on motor skills in young karate athletes aged between 8 and 12 years. METHODS: Seventy-three children, who regularly trained three karate sessions per-week were divided in two groups: high-intensity karate group (HG=53) and low-intensity karate group (LG=20). HG trained for 7 days: with two sessions per-day (one hour per-session) including specific karate techniques as well as coordination, balance, and flexibility exercises. LG, however, followed the same number of karate training sessions as before the beginning of the study (i.e., three sessions per-week with one hour per-session). Participants performed a battery of tests, 24 hours pre- and one week post-training: a medicine ball throw (MBT), standing long jump (SLJ), active joint flexibility (JM) and lateral/frontal jumps (JLT). RESULTS: Significant differences between results of pre and post karate training (ANOVA with repeated measures) included: MBT (P<0.05), SLJ (P<0.0001), JM (P<0.0001), JLT (P<0.0001); whilst the interaction training × time was: MBT (P=0.145), SLJ (P<0.0001), JM (P<0.0001), JLT (P< 0.0001). The HG significantly improved their performance on MBT by 3.23% (P<0.05), SLJ by 5.09% (P<0.001), JM by 1.51% (P<0.001), and JLT by 21.36% (P<0.001). For LG group, there were no significant differences between pre and post-testing in all fitness tests. CONCLUSION: Muscular power, flexibility and coordination represent the basics of karate fitness component. In this regard, short term high-intensity karate training represents an effective method for enhancing muscular power and range of motion (i.e. flexibility) in young karate athletes aged between 8 and 12 years. Further studies are needed to support these findings with deeper data.

Footstep manipulation during uphill running.

The present study investigated the effects of step frequency manipulation during training on slopes (2%) on biomechanical parameters at Iso-Efficiency Speed (without increasing the metabolic demand). 24 male marathon runners were randomly allocated to one of 2 training groups for 3 weeks: step frequency manipulation group (SFM, n=12) and free step frequency group (SFF, n=12). Lower limb kinematic parameters were measured before and after the 3 weeks training. The SFM group increased step length 4.30% (p<0.001), flight time 29.48% (p<0.001) and decreased contact time 14% (p<0.01). These findings coincide with characteristics of better running performances. The SFF group did not elicit such results. The results from the study could help coaches to devise training methods which could improve an athlete's performance through increasing step length. The method provided may aid faster race times for athletes.

Measurement of pulmonary gas exchange variables and lactic anaerobic capacity during field testing in elite indoor football players.

AIM: The aims of this study were: 1) to examine the gas exchange responses of elite indoor football players to a repeated sprint ability (RSA) test; and 2) to verify whether or not the excess of carbon dioxide production (CO2excess) correlates with blood lactate accumulation during RSA field testing. METHODS: Eleven elite male indoor football players were recruited. A preliminary incremental exercise test on a treadmill was performed to elicit V'O2max. Then, participants underwent an RSA test consisting in a shuttle running through a course with various changes of direction while wearing a portable gas analyzer able to provide values of oxygen uptake, carbon dioxide production, and CO2excess. BLa concentrations during recovery were also measured. RESULTS: The main results were that: 1) during the RSA test subjects did not reached the V'O2max level achieved in the preliminary test; 2) during the RSA test BLa levels were higher compared with the preliminary test; 3) the peak BLa concentration during recovery was significantly correlated with the average CO2excess CONCLUSION: It was concluded that the RSA test did not appear to be useful to elicit V'O2max. Rather, it seemed suitable to recruit subjects' lactic anaerobic capacity. Moreover, CO2excess appeared suitable for qualitatively estimate BLa accumulation during field testing.

Mindfulness-based stress reduction in cancer patients: impact on overall survival, quality of life and risk factor.

Mindfulness-based stress reduction, a complementary and alternative therapy, is able to decrease cancer-related fatigue, and stress and to improve the quality of life in cancer patients. Some studies evaluated if mindfulness-based stress reduction could improve some cardiometabolic and cancer risk factors, including systemic chemokines, growth factors, and pro-inflammatory biomarkers (e.g., C-reactive protein, Interleukin-1). In this narrative review, we highlight the pleiotropic beneficial effects of mindfulness-based stress reduction and its clinical impact on cardiovascular and cancer risk factors among patients with cancer in different stages. Moreover, improvements in the overall quality of life, sleep quality, and immune functions [changes in plasma levels of interleukin-4 (IL-4), interferon-γ (INF-γ), and interleukin-10 (IL-10)] will also be discussed. Albeit few clinical studies available in the literature, evidenced the beneficial effects of mindfulness-based stress reduction on the immune and cardiometabolic profile in cancer patients, providing important insights into the closest collaboration between psycho-oncologists, oncologists, and cardiologists.

Confirmatory validation analysis of the PROFFIT questionnaire to assess financial toxicity in cancer patients.

BACKGROUND: The Patient Reported Outcome for Fighting FInancial Toxicity (PROFFIT) questionnaire was developed to measure financial toxicity (FT) and identify its determinants. The aim of the present study was to confirm its validity in a prospective cohort of patients receiving anticancer treatment. PATIENTS AND METHODS: From March 2021 to July 2022, 221 patients were enrolled at 10 Italian centres. Selected items of the EORTC-QLQ-C30 questionnaire represented the anchors, specifically, question 28 (Q-28) on financial difficulties, and questions 29-30 measuring global health status/quality of life (HR-QOL). The study had 80% power to detect a 0.20 correlation coefficient (r) between anchors and PROFFIT-score (items 1-7, range 0-100, 100 indicating maximum FT) with bilateral alpha 0.05 and 80% power. Confirmatory factor analysis was conducted. FT determinants (items 8-16) were described. RESULTS: Median age of patients was 65 years, 116 (52.5%) were females, 96 (43.4%) had low education level. Confirmatory factor analysis confirmed goodness of fit of the PROFFIT-score. Significant partial correlation of PROFFIT-score was found with Q-28 (r = 0.51) and HR-QOL (r = -0.23). Mean (SD) PROFFIT-score at baseline was 36.5 (24.9); it was statistically significantly higher for patients living in South Italy, those with lower education level, those who were freelancer/unemployed at diagnosis and those who reported significant economic impact from the COVID-19 pandemic. Mean (SD) scores of determinants ranged from 17.6 (27.1) for item 14 (support from medical staff) to 49.0 (36.3) for item 10 (expenses for medicines or supplements). PROFFIT-score significantly increased with worsening response to determinants. CONCLUSIONS: External validation of PROFFIT-score in an independent sample of patients was successful. The instrument is now being used in clinical studies.

Uphill running at iso-efficiency speed.

The purpose of this study was to investigate the effects of slopes (0%, 2% and 7%) on temporal gait kinematics during running at iso-efficiency speed (IES). 65 male marathon runners were selected for this study. A single digital camera (210 Hz) was used to record motion; Dartfish5.5Pro was used to perform 2-dimensional (2D) video analysis and heart rate was recorded during the test. The parameters considered in this study were: step length (SL), flight time (FT), step frequency (SF), contact time (CT) and heart rate (HR). The results showed SL, FT and SF decreased as a result of the increasing treadmill gradient; SL=[(0-2%=8.38%, p<0.0001), (0-7%=23.61%, p<0.0001)]; FT=[(0-2%=8.92%, p<0.02), (0-7%=23.40%, p<0.0001)]; SF=[(0-2%=1.18%), (0-7%=4.02%, p<0.001)]. The CT and HR however increased with the increasing gradient CT=[(0-2%=9.06% p<0.0001), (0-7%=25.64%, p<0.0001)]; HR=[(0-2%=1.65%), (0-7%=3.58%)]. These results show a different trend of the footstep's kinematic parameters when running on a slope at IES. Moreover, we can calculate the optimal run speed on a slope without increasing the metabolic demand.

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation.

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

Free diffusion to and from the inner nuclear membrane of newly synthesized plasma membrane glycoproteins.

Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold-conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane.

Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D.

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.

Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues.

We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types.

Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac----liver transition.

Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS----L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts----macrocytes) and globin expression (zeta----alpha, epsilon----gamma).

Development of humanized mice for the study of hepatitis C virus infection.

The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.

In vitro differentiation and proliferation of human hematopoietic progenitors: the effects of interleukins 1 and 6 are indirectly mediated by production of granulocyte-macrophage colony-stimulating factor and interleukin 3.

The effect of recombinant human interleukin (IL)-1 and IL-6 on the differentiation and proliferation in vitro of human granulocyte-macrophage (GM) and erythroid progenitors has been investigated in either fetal bovine serum (FBS)-supplemented or FBS-deprived cultures. Sources of progenitor cells were unfractionated bone marrow cells or marrow cells depleted of adherent and/or T cells. Each interleukin was investigated either alone or in combination with GM-colony-stimulating factor (CSF), IL-3 and erythropoietin (Epo), or granulocyte (G)-CSF. In FBS-supplemented cultures of unfractionated marrow cells, IL-1 induced optimal GM colony growth and increased by 50% the number of erythroid bursts that formed in the presence of Epo. The addition to these cultures of a neutralizing anti-GM-CSF monoclonal antibody or of an anti-IL-3 serum decreased the growth of GM colonies by 80% and 40%, respectively. Under the same conditions, IL-6 had no effect on GM colony growth but increased by 90% the number of erythroid bursts. This effect was partially (40%) neutralized by addition of anti-IL-3 serum. IL-1 and IL-6 were weak stimuli, or had no effect at all, either alone or in combination with GM-CSF and IL-3 in FBS-deprived cultures or in FBS-supplemented cultures of nonadherent or nonadherent, T-cell-depleted marrow cells. IL-1 and IL-6 had no effect, either alone or in combination with IL-3, in maintaining the number of progenitor cells in short-term liquid suspension cultures. These results indicate that the actions of IL-1 and IL-6 on hematopoiesis are mainly indirect and mediated by the production of GM-CSF and/or IL-3 by accessory cells. However, neither IL-1 nor IL-6 alone is sufficient to stimulate production of growth factor(s) by accessory cells, and at least a second stimulus, provided by FBS, is also required. These data are in agreement with a multisignal model of regulation of the expression of growth factor genes.

Fluctuations of BFUe and CFUe cycling after erytrhoid perturbations: correlation with variations of pool size.

The pool size of erythroid burst-(BFUe) and colony-forming units (CFUe) has been evaluated in normal or hypoxia-induced polycythemic mice at sequential times after either transfusion or administration of purified erythropoietin (Ep). The present investigations were focused on the in vitro tritiated thymidine (3H-TdR) suicide index of the erythroid precursors in these two experimental models. After transfusion an early but transient decline of the DNA sythesis index was observed in the BFUe pool, whereas this parameter showed a later, more prolonged decrease within the CFUe population. Symmetric patterns were documented after Ep injection: an early, transient elevation of the 3H-TdR sensitivity at the BFUe level and a late, more persistent rise within the CFUe compartment. In all experiments no modification of this indes was observed within the pool of the CFUe. These fluctuations of BFUe and CFUe cycling were temporally consistent with modifications of their respective pool size, as previously reported. Thus, variations of the pool size may be at least partially mediated by the cycling activity. Furthermore, early fluctuations of BFUe proliferative rate indicate that, after erythroid perturbations, this pool may initially be sensitive to and regulated by modifications of Ep activity. Later, however, compensatory mechanisms allow the BFUe to escape from this early Ep influence, thus leading its cycling activity and pool size to return to normality and stabilize.

In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice.

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1(-) , Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/ 500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells approximately = 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250-1000 Rhodull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.

Regulation of differentiation of murine progenitor cells derived from blast cell colonies under serum-deprived conditions.

We have examined the effect of interleukin 3 (IL-3), granulocyte-macrophage (GM)-, granulocyte (G)-, and macrophage (M)-colony-stimulating factors (CSFs) on the induction of GM colonies from highly enriched murine hematopoietic progenitor cells under serum-deprived conditions. Each growth factor was tested alone or in combination with suboptimal concentrations of the others. The effect of each CSF on GM colony growth in fetal bovine serum (FBS)-supplemented cultures of unfractionated marrow cells is reported for comparison. GM-CSF induced GM colony growth in serum-deprived cultures of purified progenitor cells to the same extent as in FBS-supplemented cultures of unfractionated marrow cells. In contrast, IL-3 was only one-tenth as active in promoting the growth of enriched progenitor cells under serum-deprived conditions when compared with its effect on colony growth from unfractionated marrow. M-CSF and G-CSF were almost completely ineffective in both cases. G-CSF induction of GM colony growth from purified progenitor cells was restored by addition of suboptimal concentrations of IL-3 or GM-CSF, suggesting that either IL-3 or GM-CSF is required to observe the effect of G-CSF. Addition of G-CSF to GM-CSF-stimulated cultures did not increase the maximal number of colonies detected, indicating that these two growth factors may act on the same subset of progenitor cells. Addition of GM-CSF or IL-3 to IL-3- or GM-CSF-stimulated cultures, respectively, increased by 40% the maximal number of colonies detected, suggesting that these two factors act on at least partially separate subsets of GM progenitors. These data parallel the recent observations on the control of human GM colony formation under FBS-deprived conditions and support a model for the control of myeloid differentiation that requires the interplay of different growth factors.

Dependence for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7.

Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line, UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number of UT-7 cells (2400 +/- 3 vs. 13,800 +/- 300). This difference in the number of binding sites could be due to differences in cell surface (UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells expressed alpha-globin and higher levels of gamma-globin (5-fold) and beta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed beta- and gamma-globin genes in the two cell populations was measured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing gamma-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3).(ABSTRACT TRUNCATED AT 400 WORDS)

Early fluctuations of BFU-E pool size after transfusion or erythropoietin treatment.

The kinetics of erythroid burst-(BFU-E) and colony-forming units (CFU-E) have been evaluated in marrow and spleen of normal or polycythemic mice, respectively, after transfusion or administration or purified erythropoietin (Ep). Ep injection induces an early but temporary rise of BFU-E number, versus a later but more prolonged expansion of the CFU-E pool. Symmetric patterns are observed after transfusion, i.e., an early but transient depletion of the BFU-E population, versus a later but more persistent decrease of the CFU-E number. It is suggested that the size of the BFU-E compartment is Ep-dependent in the "early" phase after erythroid perturbation. Later on, however, compensatory mechanisms allow the BFU-E pool to "escape" from the early Ep influence, thus allowing its size to return to and stabilize in the near normal range. It is further suggested that the BFU-E may represent an early target cell of Ep stimulus.