Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.     

Characterization of normal peripheral blood lymphocyte colony-forming cells: cell cycle status, surface markers, and cellular growth requirements

We performed a series of studies to further clarify the nature of lymphocyte colony-forming cells (CFC) from normal peripheral blood. Mononuclear cells were separated into E-rosette-enriched (E+) and E- rosette-depleted (E-) populations and cultured in methylcellulose with conditioned media and irradiated mononuclear cells. Linear plating relationships were obtained with plating efficiencies of 0.26% +/- .02% (mean +/- SE) for E+ CFC and 0.18% +/- .02% for E- CFC. Cells in E+ colonies were T lymphocytes and in E- colonies were B lymphocytes as determined by cell surface marker analysis. Using the thymidine suicide technique, approximately one-half of CFC were found to be in cycle at any moment, and plating efficiencies and cell cycle status of E+ CFC were not changed by preincubation with PHA in liquid culture for 48 hr. Using antibody complement-mediated cytotoxicity, E+ CFC were found to be T101+, OKT3+, and Ia-, while E- CFC were OKT3- and Ia+. Using monocyte-depleted populations obtained by sedimentation at unit gravity, lymphocyte colony growth was absent in monocyte-depleted fractions, and optimal growth occurred with 40% monocytes in culture. In contrast to some previous studies, we find that lymphocyte CFC originate from a small, cycling population of cells bearing mature T or B lymphocyte markers. Entry into cell division, however, does not confer colony-forming capacity on lymphocytes. Monocytes are critical to growth of E+ CFC, and cultures severely depleted of monocytes would not be expected to form colonies.     

Multilineage hematopoietic growth factor interleukin 3 and direct activators of protein kinase C stimulate phosphorylation of common substrates

In order to investigate early signal transduction events in myeloid cells, the phosphosubstrates of an interleukin 3 (IL 3)-dependent cell line, FDC-P1, have been analyzed. Using synthetic diacylglycerol as a direct activator of the unique calcium-phospholipid-dependent phosphotransferase protein kinase C (PK-C) and genetically engineered homogeneous IL 3, we have demonstrated a common element to signal transduction events associated with these stimulants. One novel substrate, p68 (68,000 kd), was rapidly phosphorylated in either IL 3- or diacylglycerol-stimulated cells. The phosphorylation of p68 was dose- dependent, with both the physiological ligand and diacylglycerol inducing the same maximal level of phosphorylation. Phosphorylation of p68 occurred in a time-dependent manner analogous to previously described kinetics of PK-C subcellular redistribution in the FDC-P1 cell line. The p68 substrate was also phosphorylated in a cell-free system under conditions designed to activate PK-C. Phosphoamino acid analysis demonstrated that the p68 molecule phosphorylated in intact cells as well as in a calcium-phospho-lipid-dependent cell-free system was phosphorylated on threonine residues, not tyrosine. These data support the hypothesis that the activation of PK-C that occurs after IL 3-receptor interaction which leads to the rapid phosphorylation of cellular proteins is an important element of the signal transduction mechanism in FDC-P1 cells. We propose that phosphorylation of the p68 molecule is a physiochemical marker for the activation of PK-C in myeloid cells, in response to the growth-promoting physiological ligand.     

High frequency of N-ras activation in acute myelogenous leukemia

Using the NIH/3T3 cell transfection assay, activated cellular oncogenes have been detected in around 10% to 20% of human tumors. From a series of DNA preparations from tissues infiltrated with acute myelogenous leukemia (AML), 50% (3/6) caused transformation of NIH/3T3 cells. Thus AML appears to be the human tumor with the highest frequency of oncogenes detected by DNA transfection. In each case the oncogene involved was N-ras, a member of the ras gene family. Biologic and clinical parameters of AML patients with and without N-ras oncogenes in their tumors are discussed.     

Antihypertensive Therapy in African Americans: Findings From an Inner‐City Ambulatory Clinic

J Clin Hypertens (Greenwich). 2010;12:187–192. ^©2010 Wiley Periodicals, Inc. African Americans bear a greater burden of hypertension. Understanding prevailing epidemiologic patterns can facilitate the implementation and successful outcome of community programs. The authors assessed practice patterns of antihypertensive drug utilization and blood pressure (BP) control in a predominantly African American population in Brooklyn, NY, from January 1 to January 31, 2008. A total of 416 (53.1%) had hypertension, with a mean age of 61 years, and 267 (64%) were women. In general, 212 (50.9%) were at goal BP and 59.9% of those at goal were taking at least 2 drugs. Patient age correlated with the number of drugs used (r=0.14; P=.004). Patients taking β‐blockers and calcium channel blockers were older: 63.6 vs 60.1 years (P=.01) and 62.7 vs 60.3 years (P=.07), respectively. The pattern of antihypertensive use was as follows: angiotensin‐converting enzyme inhibitors, 194 (46.6%); calcium channel blockers, 162 (38.9%); diuretics, 162 (38.9%); β‐blockers, 133(32%); and angiotensin receptor blockers, 93 (22.4%). The findings of age associated with the class of medications used and a predominance of angiotensin‐converting enzyme inhibitors usage highlight possible gaps in appropriateness of antihypertensive therapy. The application of age‐appropriate race‐based antihypertensive therapy might improve BP control rates. These results strengthen arguments for investing in community‐based programs to overcome possible provider‐related and local health system barriers to achieving BP control goals.

Hypertension is the most common primary diagnosis in America and is responsible for 35.7 million office visits per year. ¹ Overall, 1 in 3 American adults have high blood pressure (BP), while 2 in 5 African American adults have high BP. ² African Americans bear a greater burden of disease, ^{3 , 4} have a variable response to conventional antihypertensive medications, ^{5 , 6} and develop more severe end‐organ damage. ^{7 , 8} In addition to pharmacologic therapy and lifestyle modifications, community‐based interventions focusing on African Americans may improve outcomes. In this light, the Centers for Disease Control and Prevention (CDC) has initiated the Racial and Ethnic Approaches to Community Health Across the US (REACH US) program. ^{9 , 10 , 11 , 12} Similarly, the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) ¹³ recommends a 3‐pronged approach of pharmacologic therapy, lifestyle changes, and public health initiatives involving community participation and mobilization. Pharmacologic therapy remains a primary focus of care, however, as it prevents and reverses end‐organ damage and improves cardiovascular outcomes. ^{14 , 15} Correspondingly, to ensure a cost‐effective, sustainable, and successful community‐based intervention, the intensity and design of public health programs involving grassroots initiatives ought to be guided by a clear understanding of prevailing epidemiologic patterns of hypertension.As such, the CDC calls for continuous surveillance of health status in minority communities so that culturally sensitive prevention strategies can be tailored to these communities and program interventions evaluated. ¹⁶ Knowledge of existing prescribing patterns, antihypertensive drug utilization, and BP control rates in the index community can provide useful information for establishing community programs to combat hypertension and gauge their effectiveness. We assessed practice patterns and BP control in a predominantly African American population serviced by a community health clinic affiliated with an internal medicine residency program.     

Dopamine increases renal oxygenation: a clinical study in post‐cardiac surgery patients

Background: Imbalance of the renal medullary oxygen supply/demand relationship can cause ischaemic acute renal failure (ARF). The use of dopamine for prevention/treatment of ischaemic ARF has been questioned. It has been suggested that dopamine may increase renal oxygen consumption (RVO₂) due to increased solute delivery to tubular cells, which may jeopardise renal oxygenation. Information on the effects of dopamine on renal perfusion, filtration and oxygenation in man is, however, lacking. We evaluated the effects of dopamine on renal blood flow (RBF), glomerular filtration rate (GFR), RVO₂ and renal O₂ demand/supply relationship, i.e. renal oxygen extraction (RO₂Ex). Methods: Twelve uncomplicated, mechanically ventilated and sedated post‐cardiac surgery patients with pre‐operatively normal renal function were studied. Dopamine was sequentially infused at 2 and 4 ug/kg/min. Systemic haemodynamics were evaluated by a pulmonary artery catheter. Absolute RBF was measured using two independent techniques: by the renal vein thermodilution technique and by infusion clearance of paraaminohippuric acid (PAH), with a correction for renal extraction of PAH. The filtration fraction (FF) was measured by the renal extraction of ⁵¹Cr‐EDTA. Results: Neither GFR, tubular sodium reabsorption nor RVO₂ was affected by dopamine, which increased RBF (45–55%) with both methods, decreased renal vascular resistance (30–35%), FF (21–26%) and RO₂Ex (28–34%). The RBF/CI ratio increased with dopamine. Dopamine decreased renal PAH extraction, suggestive of a flow distribution to the medulla. Conclusions: In post‐cardiac surgery patients, dopamine increases the renal oxygenation by a pronounced renal pre‐and post‐glomerular vasodilation with no increases in GFR, tubular sodium reabsorption or renal oxygen consumption.     

Plitidepsin has a dual effect inhibiting cell cycle and inducing apoptosis via Rac1/c-Jun NH2-terminal kinase activation in human melanoma cells

Melanoma is the most aggressive skin cancer and a serious health problem worldwide because of its increasing incidence and the lack of satisfactory chemotherapy for late stages of the disease. The marine depsipeptide Aplidin (plitidepsin) is an antitumoral agent under phase II clinical development against several neoplasias, including melanoma. We report that plitidepsin has a dual effect on the human SK-MEL-28 and UACC-257 melanoma cell lines; at low concentrations (相似文献