Protection of glucotoxicity by a tripeptide derivative of α-melanocyte-stimulating hormone in human epidermal keratinocytes.

BACKGROUND: Chronic skin ulcers are a major complication and a therapeutic challenge in patients with diabetes mellitus. Glucose-induced accumulation of reactive oxygen species (ROS) is considered to be an important pathogenetic factor in diabetes. OBJECTIVES: To characterize the impact of high glucose (HG) on normal human keratinocytes (NHKs) and examine if Lys-d-Pro-Thr (KdPT), a tripeptide derived from α-melanocyte-stimulating-hormone, has protective effects. METHODS: We investigated the key functions of NHKs under HG conditions with or without KdPT in vitro as well as ex vivo employing a skin organ culture model. RESULTS: HG impaired metabolic activity, cell proliferation, viability and migration of NHKs. As shown by atomic force microscopy HG altered the biophysical properties of NHKs, i.e. cell size and elasticity. Glucotoxicity in NHKs was paralleled by the induction of intracellular ROS and endoplasmic reticulum stress. KdPT attenuated HG-induced oxidative stress and antagonized the effects of glucose on cell viability, metabolic activity and migration. Importantly, KdPT also antagonized the suppressive effect of HG on epidermal migration in wounded human skin organ cultures. CONCLUSIONS: Our findings highlight a novel effect of KdPT that could be exploited for the future therapy of diabetic skin ulcers.

Oral contraceptive progestins and angiotensin-dependent control of the renal circulation in humans.

Oral contraceptive (OC) use is associated with increased intrarenal renin-angiotensin-aldosterone system (RAA System) activity and risk of nephropathy, though the contribution of progestins contained in the OC in the regulation of angiotensin-dependent control of the renal circulation has not been elucidated. A total of 18 OC users (8 non-diabetic, 10 Type 1 diabetic) were studied in high salt balance, a state of maximal RAA System suppression. Progestational and androgenic activity of the progestin in each OC was standardized to that of the reference progestin norethindrone. Renal plasma flow (RPF) was measured by para-aminohippurate clearance at baseline and in response to angiotensin-converting enzyme (ACE) inhibition. There was a positive correlation between OC progestational activity and the RPF response to ACE inhibition (r=0.52, P=0.03). Similar results were noted with OC androgenic activity (r=0.54, P=0.02). On subgroup analysis, only non-diabetic subjects showed an association between progestational activity and angiotensin-dependent control of the renal circulation (r=0.71, P=0.05 non-diabetic; r=0.14, P=0.7 diabetic; P=0.07 between groups). Similar results were noted with respect to androgenic activity (r=0.88, P=0.005 non-diabetic; r=-0.33, P=0.3 diabetic; P=0.002 between groups). Our results suggest that the OC progestin component is a significant influence on the degree of angiotensin-dependent control of the renal circulation, though these findings may not apply to women with diabetes.

Body-mass index, waist-size, waist-hip ratio and cardiovascular risk factors in urban subejcts.

BACKGROUND AND OBJECTIVE: Influence of obesity as determinant of cardiovascular risk factors has not been well studied. To determine association of obesity, measured by body-mass index (BMI), waist-size or waist-hip ratio (WHR), with multiple risk factors in an urban Indian population we performed an epidemiological study. METHODS: Randomly selected adults > or = 20 years were studied using stratified sampling. Target sample was 1800 (men 960, women 840). 1123 subjects (response 62.4%) were evaluated and blood samples were available in 532 men and 559 women (n=1091, response 60.6%). Measurement of anthropometric variables, blood pressure, fasting blood glucose and lipids was performed. Atherosclerosis risk factors were determined using current guidelines. Pearson's correlation coefficients (r) of BMI, waist and WHR with various risk factors were determined. BMI was categorized into five groups: <20.0 Kg/m2, 20.0-22.9, 23.0-24.9, 25.0-29.9, and > or = 30 Kg/m2; waist size was divided into five groups and WHR into six groups in both men and women. Prevalence of cardiovascular risk factors, smoking, hypertension, diabetes, metabolic syndrome and dyslipidaemias was determined in each group and trends analyzed using least-squares regression. RESULTS: There is a significant positive correlation of BMI, waist-size and WHR with systolic BP (r= 0.46 to 0.13), diastolic BP (0.42 to 0.16), fasting glucose (0.15 to 0.26), and LDL cholesterol (0.16 to 0.03) and negative correlation with physical activity and HDL cholesterol (-0.22 to -0.08) in both men and women (p<0.01). With increasing BMI, waist-size and WHR, prevalence of hypertension, diabetes, and metabolic syndrome increased significantly (p for trend <0.05). WHR increase also correlated significantly with prevalence of high total and LDL cholesterol and triglycerides (p <0.05). CONCLUSIONS: There is a continuous positive relationship of all markers of obesity (body-mass index, waist size and waist hip ratio) with major coronary risk factors- hypertension, diabetes and metabolic syndrome while WHR also correlates with lipid abnormalities.

Internationally distributed frozen oyster meat causing multiple outbreaks of norovirus infection in Australia.

BACKGROUND: Between November 2003 and January 2004, outbreaks of norovirus in 3 Australian jurisdictions involving 83 cases of illness were associated with imported oyster meat. METHODS: Cohort studies were conducted in 2 jurisdictions to identify relative risks of illness for the consumption of oysters. A case series was conducted in the third jurisdiction. RESULTS: The cohort studies conducted in the first 2 jurisdictions identified relative risks of illness of 17 (95% confidence interval, 5-51) and 35 (95% confidence interval, 5-243), respectively, for the consumption of oysters. Multiple strains of norovirus were detected in fecal specimens from 8 of 14 patients and in 1 of the 3 batches of implicated oyster meat using seminested reverse-transcriptase polymerase chain reaction methods. Traceback investigations revealed that all oyster meat was harvested from the same estuary system in Japan within the same month. CONCLUSIONS: These outbreaks demonstrate the potential of foodborne disease to spread internationally and the need for national and international collaboration to investigate such outbreaks. Foodborne illness related to norovirus is underestimated because of underreporting of human cases and challenges in laboratory detection of viruses in foods, both of which can delay public health action.

Fasting glucose and cardiovascular risk factors in an urban population.

OBJECTIVE: To test the hypothesis that blood glucose levels in the range of normoglycemia are associated with increased cardiovascular risk we performed an epidemiological study in an urban population. METHODS: Randomly selected adults > or = 20 years were studied using stratified sampling. Target sample was 1800 (men 960, women 840) of which 1123 subjects participated. Blood samples were available in 1091 subjects (60.6%, men 532, women 559). Measurement of anthropometric variables, blood pressure, fasting blood glucose and lipids was performed. Cardiovascular risk factors were determined using US Adult Treatment Panel-3 guidelines. Pearson's correlation coefficients (r) of fasting glucose with various risk factors were determined. Fasting glucose levels were classified into various groups as < 75 mg/dl, 75-89 mg/dl, 90-109 mg/dl, 110-125 mg/dl and > 126 mg/dl or known diabetes. Prevalence of cardiovascular risk factors was determined in each group. RESULTS: There was a significant positive correlation of fasting glucose in men and women with body mass index (r = 0.20, 0.12), waist-hip ratio (0.17, 0.09), systolic blood pressure (0.07, 0.22), total cholesterol (0.21, 0.15) and triglycerides (0.21, 0.25). Prevalence (%) of cardiovascular risk factors in men and women was smoking/tobacco use in 37.6 and 11.6, hypertension in 37.0 and 37.6, overweight and obesity in 37.8 and 50.3, truncal obesity in 57.3 and 68.0, high cholesterol > or = 200 mg/dl in 37.4 and 45.8, high triglycerides > or = 150 mg/dl in 32.3 and 28.6 and metabolic syndrome in 22.9 and 31.6 percent. In various groups of fasting glucose there was an increasing trend in prevalence of overweight/obesity, hypertension, hypercholesterolaemia, hypertriglyceridaemia, and metabolic syndrome (Mantel-Haenzel X2 for trend, p < 0.05) and fasting glucose < 75 mg/dl was associated with the lowest prevalence of these risk factors. CONCLUSIONS: There is a continuous relationship of fasting glucose levels with many cardiovascular risk factors and level < 75 mg/dl is associated with the lowest prevalence.

Serial epidemiological surveys in an urban Indian population demonstrate increasing coronary risk factors among the lower socioeconomic strata.

OBJECTIVE: To determine trends of coronary risk factors in an Indian urban population and their association with educational level as marker of socioeconomic status. METHODS: Two successive coronary risk factor surveys were performed in randomly selected individuals. In the first study (in 1995) 2212 subjects (1415 men, 797 women) and in the second (in 2002) 1123 subjects (550 men, 573 women) were studied. Details of smoking, physical activity, hypertension, diabetes, coronary heart disease, body-mass index, waist-hip ratio, blood pressure and electrocardiography were evaluated. Fasting blood was examined for lipid levels in 297 (199 men, 98 women) in the first and in 1082 (532 men, 550 women) in the second study. Educational status was classified into Group 0: no formal education, Group I: 1-10 years, Group II: 11-15 years, and Group III: > 16 years. Current definitions were used for risk factors in both the studies. RESULTS: Prevalence of coronary risk factors, adjusted for age and educational status, in the first and second study in men was smoking/tobacco in 38.7 vs. 40.5%, leisure time physical inactivity in 70.8 vs. 66.1%, hypertension (> or = 140 and/or 90 mm Hg) in 29.5 vs. 33.7%, diabetes history in 1.1 vs. 7.8%, obesity (body-mass index > or = 25 Kg/m2) in 20.7 vs. 33.0%, and truncal obesity (waist:hip > 0.9) in 54.7 vs. 54.4%. In women, tobacco use was in 18.7 vs. 20.5%, leisure time physical inactivity in 72.4 vs. 75.3%, hypertension in 36.9 vs. 33.7%, diabetes history in 1.0 vs. 7.3%, obesity in 19.9 vs. 39.4%, and truncal obesity (waist:hip > 0.8) in 70.1 vs. 69.2%. In men, high total cholesterol > or = 200 mg/dl was in 24.6 vs. 37.4%, high LDL cholesterol > or = 130 mg/dl in 22.1 vs. 37.0%, high triglycerides > or = 150 mg/dl in 26.6 vs. 30.6% and low HDL cholesterol < 40 mg/dl in 43.2 vs. 54.9%; while in women these were in 22.5 vs. 43.1%, 28.6 vs. 45.1%, 28.6 vs. 28.7% and 45.9 vs. 54.2% respectively. In the second study there was a significant increase in diabetes, obesity, hypertension (men), total- and LDL cholesterol and triglycerides and decrease in HDL cholesterol (p < 0.05). In the first study with increasing educational status a significant increase of obesity, total cholesterol, LDL cholesterol and triglycerides and decrease in smoking was observed. In the second study increasing education was associated with decrease in smoking, leisure-time physical inactivity, total and LDL cholesterol, and triglycerides and increase in obesity, truncal obesity and hypertension (Least-squares regression p < 0.05). Increase in smoking, diabetes and dyslipidaemias was greater in the less educated groups. CONCLUSIONS: Significant increase in coronary risk factors--obesity, diabetes, total-, LDL-, and low HDL cholesterol, and triglycerides is seen in this urban Indian population over a seven year period. Smoking, diabetes and dyslipidaemias increased more in low educational status groups.

Thyroid hormone receptor-interacting protein 1 modulates cytokine and nuclear hormone signaling in erythroid cells.

Erythropoietin (Epo) and thyroid hormone (T(3)) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T(3) receptor, providing a link between the Epo and T(3) signaling pathways. Trip-1 co-localized with Lyn and the T(3) receptor alpha in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T(3) stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T(3) reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27(Kip1) and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T(3)-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27(Kip1), and blocking T(3) response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

Low dose intrathecal ropivacaine with or without sufentanil provides effective analgesia and does not impair motor strength during labour: a pilot study.

PURPOSE: Although ropivacaine has been used to provide spinal anesthesia in the surgical population, its intrathecal administration for labour analgesia has only recently been described. We evaluated the effects of low dose intrathecal ropivacaine with or without sufentanil for labour analgesia. METHODS: Thirty-six term parturients in active labour were randomly assigned to receive 3 mg of intrathecal ropivacaine (group R) or 3 mg ropivacaine with 10 microg of sufentanil (group RS). Patients were evaluated by a blinded observer for hypotension, linear analogue score (VAS 0-100) for labour pain, motor power in the lower limbs, onset of analgesia, sensation to cold and pin prick, duration of analgesia, and neonatal Apgar scores. The following day patients were assessed for satisfaction, headache and neurologic deficit. RESULTS: The mean duration of analgesia in the R group was 41.4 +/- 4.9 min and 95.0 +/- 6.1 min in the RS group (mean +/- SEM, P=0.0001). All subjects had satisfactory analgesia at five minutes, although analgesia from the ropivacaine- sufentanil combination was superior to that provided by ropivacaine alone. Total duration of labour was no different between the groups (R- 306 +/- 34, RS- 384 +/- 44 min, P=0.17). No patient showed evidence of motor block. All patients were satisfied with the labour analgesia. No neurological complications were observed. CONCLUSIONS: Low dose ropivacaine provides effective analgesia during labour via the intrathecal route. It can be mixed with sufentanil in the above-mentioned concentrations to improve both the quality and duration of analgesia. Fetal outcome remains favourable. It may provide minimal or no motor block, to facilitate ambulation.

Lipid abnormalities in coronary heart disease: a population-based case-control study.

BACKGROUND: We performed a case-control study to estimate lipid-cholesterol fractions in patients with coronary heart disease and compared them with population-based controls. METHODS AND RESULTS: A total of 635 newly diagnosed patients with coronary heart disease (518 males and 117 females) and 632 subjects (346 males and 286 females) obtained from an ongoing urban coronary heart disease risk factor epidemiological study were evaluated. Age-specific lipid values (total cholesterol, low-density lipoprotein, high-density lipoprotein, triglycerides, and total:high-density lipoprotein cholesterol ratio) were compared using the t-test. Age-adjusted prevalence of dyslipidemia as defined by the US National Cholesterol Education Program was compared using the Chi-square test. In all the age groups, and in both males and females, levels of total and low-density lipoprotein cholesterol were not significantly different. In males, the high-density lipoprotein cholesterol (mg/dl) was significantly lower in patients with coronary heart disease as compared to controls in the age groups 30-39 years (35.1+/-11 v. 43.7+/-9), 40-49 years (39.0+/-10 v. 47.1+/-8), 50-59 years (38.9+/-11 v. 43.8+/-9) and 60-69 years (38.6+/-11, v. 42.8+/-7) (p<0.05). In females, high-density lipoprotein cholesterol was less in the age groups 30-39 years (30.2+/-9 v. 40.7+/-9), 50-59 years (39.7+/-12 v. 44.7+/-8) and 60-69 years (35.6+/-11 v. 42.2+/-9). The level of triglycerides was significantly higher in male patients in the age groups 40-49 years (195.3+/-96 v. 152.8+/-78), 50-59 years (176.7+/-76 v. 162.9+/-97), 60-69 years (175.5+/-93 v. 148.1+/-65) and >70 years (159.8+/-62 v. 100.0+/-22); and in female patients in the age group 30-39 years (170.8+/-20 v. 149.9+/-9) (p<0.05). The total:high-density lipoprotein cholesterol ratio was significantly higher in all age groups in male as well as female patients with coronary heart disease (p<0.05). CONCLUSIONS: An age-adjusted case-control comparison showed that the prevalence of hypertension, diabetes, high total cholesterol (> or =200 mg/dl) (males 48.8% v. 20.2%; females 59.8% v. 33.4%) and high low-density lipoprotein cholesterol (> or =130 mg/dl) (males 42.1% v. 15.0%; females 52.1% v. 31.0%) was significantly more in cases than in controls. The prevalence of low high-density lipoprotein cholesterol (<35 mg/dl) (males 39.6% v. 6.2%; females 39.3% iv 9.5%), high total:high-density lipoprotein ratio (> or = 5.0) and high triglycerides (> or =200 mg/ dl: males 39.6%, v. 10.2%; females 17.1% v. 11.9%) was also significantly higher in cases (p<0.05).

Predictors of breakthrough pain during labor epidural analgesia.

UNLABELLED: Parturients who receive labor epidural analgesia may experience breakthrough pain that requires supplemental medications. We investigated the factors associated with breakthrough pain. This prospective observational study included 1963 parturients who received epidural analgesia. Subjects were categorized into two groups on the basis of the number of episodes of breakthrough pain: the Recurrent Breakthrough Pain (RBP) group experienced three or more episodes. Univariate and multivariate regression analyses were used to evaluate factors associated with the RBP group. By multivariate analysis, nulliparity, heavier fetal weight, and epidural catheter placement at an earlier cervical dilation were found to be independently associated with the RBP group. These factors may predict which parturients' analgesia may be complicated by breakthrough pain. Parturients who received a combined spinal/epidural technique were less likely to be associated with the RBP group. The combined spinal/epidural technique may be superior to conventional epidural anesthesia, because breakthrough pain occurred less often. It is interesting to note that the characteristics that are associated with the RBP group are similar to those that have been associated with increased severity of maternal pain. IMPLICATIONS: Nulliparity, heavier fetal weight, and epidural catheter placement at an early cervical dilation are predictors of breakthrough pain during epidural labor analgesia. The combined spinal/epidural technique may be associated with a decreased incidence of breakthrough pain.

Maturation of erythroid cells and erythroleukemia development are affected by the kinase activity of Lyn.

This study examined the impact of the tyrosine kinase Lyn on erythropoietin-induced intracellular signaling in erythroid cells. In J2E erythroleukemic cells, Lyn coimmunoprecipitated with numerous proteins, including SHP-1, SHP-2, ras-GTPase-activating protein, signal transducers and activators of transcription (STAT) 5a, STAT5b, and mitogen-activated protein kinase; however, introduction of a dominant-negative Lyn (Y397F Lyn) inhibited the interaction of Lyn with all of these molecules except SHP-1. Cells containing the dominant-negative Lyn displayed altered intracellular phosphorylation patterns, including mitogen-actiated protein kinase, but not erythropoietin receptor, Janus-activated kinase (JAK) 2, or STAT5. As a consequence, erythropoietin-initiated differentiation and basal proliferation were severely impaired. Y397F Lyn reduced the protein levels of erythroid transcription factors erythroid Kruppel-like factor and GATA-1 up to 90%, which accounts for the inability of J2E cells expressing Y397F Lyn to synthesize hemoglobin. Although Lyn was shown to bind several sites on the cytoplasmic domain of the erythropoietin receptor, it was not activated when a receptor mutated at the JAK2 binding site was ectopically expressed in J2E cells indicating that JAK2 is the primary kinase in erythropoietin signaling and that Lyn is a secondary kinase. In normal erythroid progenitors, erythropoietin enhanced phosphorylation of Lyn; moreover, exogenous Lyn increased colony forming unit-erythroid, but not burst forming uniterythroid, colonies from normal progenitors, demonstrating a stage-specific effect of the kinase. Significantly, altering Lyn activity in J2E cells had a profound effect on the development of erythroleukemias in vivo: the mortality rate was markedly reduced and latent period extended when either wild-type Lyn or Y397F Lyn was introduced into these cells. Taken together, these data show that Lyn plays an important role in intracellular signaling in nontransformed and leukemic erythroid cells.

Ectopic expression of transcription factor NF-E2 alters the phenotype of erythroid and monoblastoid cells.

In this study, regulation of transcription factor NF-E2 was examined in differentiating erythroid and myeloid cells, and the impact of raising NF-E2 concentrations within these cell types was assessed. NF-E2 was expressed in the J2E erythroid cell line, but the levels increased only marginally during erythropoietin-induced differentiation. In contrast, rare myeloid variants of J2E cells did not express NF-E2. Although NF-E2 was present in M1 monoblastoid cells, it was undetectable as these cells matured into macrophages. Compared with erythroid cells, transcription of the NF-E2 gene was reduced, and the half-life of the mRNA was significantly shorter in monocytoid cells. Ectopic expression of NF-E2 had a profound impact upon the J2E cells; morphologically mature erythroid cells spontaneously emerged in culture, but the cells failed to synthesize hemoglobin, even in the presence of erythropoietin. Although proliferation and viability increased in the NF-E2-transfected J2E cells, their responsiveness to erythropoietin was severely diminished. Strikingly, increasing the expression of NF-E2 in M1 cells produced sublines that contained erythroid or immature megakaryocytic cells. Finally, overexpression of NF-E2 in primary hemopoietic progenitors from fetal liver increased erythroid colony formation in the absence of erythropoietin. These data demonstrate that elevated NF-E2 (i) had a dominant effect on the phenotype and maturation of J2E erythroid cells, (ii) was able to reprogram the M1 monocytoid line, and (iii) promoted the development of erythroid colonies by normal progenitors.

An association between severe labor pain and cesarean delivery.

UNLABELLED: The relationship between epidural analgesia and cesarean delivery remains controversial. Several studies have documented an association, although others have not. This inconsistency may result from an association between severe labor pain and dystocia. We hypothesized that dystocia causes severe labor pain, such that more epidural medication is required to maintain comfort. We examined the relationship between labor outcome and severe pain, defined by the number of supplemental epidural boluses. We retrospectively reviewed the anesthesia records of 4493 parturients who received small-dose labor epidural analgesia. An independent association was found between operative delivery and maternal age, body mass index, nulliparity, fetal weight, induction of labor, and the number of boluses required during labor. By using multivariate analysis, the odds ratio of cesarean delivery among women who required at least three boluses was 2.3 compared with those who required two boluses or less. No association was found between the concentration of bupivacaine in the epidural infusion and operative delivery. Because women with cesarean deliveries appeared to have more pain, degree of labor pain may be a confounding factor in studies examining epidural analgesia and outcome. IMPLICATIONS: This is a retrospective observational study demonstrating an association between labor pain and cesarean delivery. Our results provide an alternative explanation of why epidural analgesia is associated with cesarean delivery.

Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia development in vivo.

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Delta257 and Delta321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Delta321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Delta321 receptor, the Delta257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias. Oncogene (2000) 19, 953 - 960.

HS1 interacts with Lyn and is critical for erythropoietin-induced differentiation of erythroid cells.

Erythroid cells terminally differentiate in response to erythropoietin binding its cognate receptor. Previously we have shown that the tyrosine kinase Lyn associates with the erythropoietin receptor and is essential for hemoglobin synthesis in three erythroleukemic cell lines. To understand Lyn signaling events in erythroid cells, the yeast two-hybrid system was used to analyze interactions with other proteins. Here we show that the hemopoietic-specific protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E erythroleukemic cells to determine the impact upon responsiveness to erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E line-the cells were smaller, more basophilic than the parental proerythoblastoid cells and had fewer surface erythropoietin receptors. Moreover, basal and erythropoietin-induced proliferation and differentiation were markedly suppressed. The inability of cells containing the truncated HS1 to differentiate may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin stimulation of these cells resulted in rapid, endosome-mediated degradation of endogenous HS1. The truncated HS1 also suppressed the development of erythroid colonies from fetal liver cells. These data show that disrupting HS1 has profoundly influenced the ability of erythroid cells to terminally differentiate.

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit.

We report here the isolation of a new member of the ADP-ribosylation factor (ARF)-like family (ARL-6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage-restricted expression, ARL-6 mRNA increased with erythropoietin-induced maturation of J2E cells, and decreased with interleukin 6-induced differentiation of M1 monoblastoid cells. In tissues, ARL-6 mRNA was most abundant in brain and kidney. While ARL-6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP-gammaS, like many members of the ARF/ARL family. Using the yeast two-hybrid system, six molecules which interact with ARL-6 were identified including SEC61beta, a subunit of the heterotrimeric protein conducting channel SEC61p. Co-immunoprecipitation of ARL-6 confirmed a stable association between ARL-6 and SEC61beta in COS cells. These results demonstrate that ARL-6, a novel member of the ADP-ribosylation factor-like family, interacts with the SEC61beta subunit.

Transcription factor erythroid Krüppel-like factor (EKLF) is essential for the erythropoietin-induced hemoglobin production but not for proliferation, viability, or morphological maturation.

The erythroid Krüppel-like factor (EKLF) is essential for the transcription of betamaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.

Intravenous oxytocin in patients undergoing elective cesarean section.

The objective of this study was to compare four different doses of oxytocin to determine its minimal effective dose during elective cesarean section. A prospective, double-blind, randomized study was undertaken in 40 healthy term parturients presenting for elective cesarean section under regional anesthesia. Subjects were assigned to one of four groups. Group I received 5 IU, Group II 10 IU, Group III 15 IU, and Group IV 20 IU of oxytocin after clamping of the umbilical cord. Uterine tone was assessed by palpation on a linear analog scale (LAS) of 0 to 10 (0 = completely atonic, 10 = fully contracted) at 5, 10, 15 and 20 min after the start of oxytocin infusion. Estimated blood loss (EBL) and the difference in pre- and postoperative hematocrit (delta Hct) were also recorded. At alpha = 0.05, the study design had a power of 95% to detect a 25% difference in the LAS between the four groups. There were no differences in the uterine tone in the four groups at any of the four intervals. EBL and delta Hct were similar in all four groups. There appears to be no benefit in terms of degree of uterine contraction and amount of blood loss to administering more than 5 IU of intravenous oxytocin to term parturients undergoing elective cesarean section with a neuraxial block.

Prevalence of coagulation abnormalities associated with intrauterine fetal death.

PURPOSE: The purpose of this study was to determine factors associated with abnormal coagulation in the setting of intrauterine fetal death (IUFD). METHODS: We reviewed the charts of 238 patients diagnosed with IUFD over ten years. Data included demographics, co-existing obstetric disease and coagulation studies. A coagulation score was assigned based on the platelet count, prothrombin time, activated partial thromboplastin time and plasma fibrinogen concentration. Approximately 90% of the study population had coagulation scores < 4. A score of > or = 4 was considered abnormal. RESULTS: Complete coagulation analysis was available in 183/238 patients (77%) within 24 hr of delivery. One hundred and sixty-four of these (89.6%) had a coagulation score, < 4 and 19 had a score > or = 4 (10.4%). No relationship between the coagulation score and age, parity, gestational age at delivery, and number of days the dead fetus remained in utero was found. A coagulation score > = or 4 was associated with the presence of a pregnancy-related disease (P < 0.05), notably abruption (P < 0.001) and uterine perforation (P < 0.05). Four patients without co-existing disease (3.2%), had a coagulation score > or = 4. CONCLUSION: In most pregnancies complicated by fetal demise, the fetus and placenta are delivered within one week of fetal demise. The previously reported severe coagulation disturbances are largely eliminated by early delivery. Our study shows that coagulation abnormalities occur in some patients with uncomplicated IUFDs (3.2%) and that this number rises in the presence of abruption or uterine perforation.