Site-directed mutagenesis of platelet glycoprotein Ib alpha demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279

The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIb alpha is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIb alpha protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.     

Increased p21(CIP1/WAF1) and B cell lymphoma leukemia-x(L) expression and reduced apoptosis in alveolar macrophages from smokers

Alveolar macrophages (AMs) are the predominant defense cells in the airway, and their numbers are increased in smokers and subjects with chronic obstructive pulmonary disease. This increase may result from increased recruitment, increased proliferation, or reduced cell death. Apoptosis regulates inflammatory cell survival, and p21(CIP1/WAF1) is an important inhibitory regulator of cycle progression after oxidative stress. We have investigated whether chronic smoke exposure influences the expression and localization of cell cycle and apoptotic proteins in AM and bronchial epithelial cells in vivo. The increased numbers of AMs seen in smokers were only partially due to enhanced proliferation. p21(CIP1/WAF1) protein expression was increased in AMs and biopsies isolated from smokers and was found predominantly within the cytoplasm. In addition, B cell lymphoma leukemia (Bcl)-x(L), an antiapoptotic regulator, was also highly expressed in macrophages from smokers compared with nonsmokers and subjects with asthma. Hydrogen peroxide, an oxidative stress, induced cytoplasmic expression of p21(CIP1/WAF1) and failed to induce apoptosis in an in vitro model. These results suggested that AM and bronchial epithelial cells from smokers, in contrast to those from normal subjects and subjects with asthma, have reduced cell death. Thus, oxidative stress induced by cigarette smoking may contribute to the chronicity of inflammation in the airway, through a reduction of apoptosis.     

An interplay of genetic and environmental factors in familial hepatitis and myasthenia gravis

A family is described in which there occurred two cases of the lupoid type of active chronic hepatitis with cirrhosis, one of chronic persistent hepatitis, and one of myasthenia gravis. The two cases of lupoid hepatitis were in the proposita, a schoolgirl aged 16 years, and her great-aunt aged 69 years whom she had never met. The case of myasthenia gravis was that of the father. The whole family, except the great-aunt, had been exposed to an epidemic of infectious hepatitis five years previously, and the girl and her brother had contracted this disease. The schoolgirl later developed active chronic hepatitis while her brother had chronic persistent hepatitis without immunological concomitants.APART FROM COINCIDENCE, SOME COMBINATION OF THREE PROCESSES WAS REQUIRED TO ACCOUNT FOR THE ILLNESSES IN THIS FAMILY: a genetic predisposition to chronic liver disease in particular, a genetic predisposition to autoimmune reactions in general, and a ;triggering' effect of infection with the hepatitis virus.     

Antibody to two forms of dihydrolipoamide acetyltransferase (PDC-E2) in primary biliary cirrhosis

ABSTRACT— Dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), is the major autoantigen in primary biliary cirrhosis. By immunoblotting with sera from patients with primary biliary cirrhosis, we observed a double band, of molecular weight 70 and 74 kD for PDC-E2, when a preparation of bovine heart mitochondria was not boiled prior to electrophoresis. This double band could also be detected using antisera raised in rats or rabbits against intact PDC or PDC-E2, but not in antisera raised against a synthetic decamer representing the lipoic acid binding sequence of PDC-E2; the latter reacted only with the 74 kD component. Antibody eluted from either the 70 or 74 kD component reacted with both 70 and 74 kD components. By ELISA, sera from patients with primary biliary cirrhosis reacted more strongly with a non-boiled than a boiled PDC-E2, whereas immune animal sera reacted equally with both preparations. Thus, according to whether preparations of PDC are boiled or not, two conformationally alternative forms of the PDC-E2 protein can be revealed by immunoblotting. The two forms in non-boiled preparations migrate at molecular weights corresponding to 70 and 74 kD.     

Selective surgery for peptic ulcer: a review

Peptic ulcer patients do not fall into one secretory pattern and methods are available by which the secretory pattern may be assessed. Provided this assessment is accurate and the operation appropriate to the requirements of the individual patient is carried out, the side-effects are not severe.

The mortality from gastric surgery need not be above 1% and even when resection is involved the technical hazards of the operation are rarely the cause of death in experienced hands.

Further improvement in immediate and long-term results and protection against recurrence may be expected as the principles of selection become better understood and surgical procedures adequate to each requirement become standardized.

     

Role of adenosine receptors in regulating chemotaxis and cytokine production of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) are potent regulators of immune function and the major source of type I interferon (IFN) following viral infection. PDCs are found at sites of inflammation in allergic reactions, autoimmune disorders, and cancer, but the mechanisms leading to the recruitment of PDCs to these sites remain elusive. During inflammation, adenosine is released and functions as a signaling molecule via adenosine receptors. This study analyzes adenosine receptor expression and function in human PDCs. Adenosine was found to be a potent chemotactic stimulus for immature PDCs via an A(1) receptor-mediated mechanism. The migratory response toward adenosine was comparable to that seen with CXCL12 (stromal-derived factor-1 alpha [SDF-1 alpha), the most potent chemotactic stimulus identified thus far for immature PDCs. Upon maturation, PDCs down-regulate the A(1) receptor, resulting in a loss of migratory function. In contrast, mature PDCs up-regulate the A(2a) receptor, which is positively coupled to adenylyl cyclase and has been implicated in the down-regulation of DC cytokine-producing capacity. We show that in mature PDCs adenosine reduces interleukin-6 (IL-6), IL-12, and IFN-alpha production in response to CpG oligodeoxynucleotides (ODN). These findings indicate that adenosine may play a dual role in PDC-mediated immunity by initially recruiting immature PDCs to sites of inflammation and by subsequently limiting the extent of the inflammatory response induced by mature PDCs by inhibiting their cytokine-producing capacity.     

The effects of carbon dioxide on oxygenation and systemic, cerebral, and pulmonary vascular hemodynamics after the bidirectional superior cavopulmonary anastomosis

OBJECTIVES: We investigated the effects of different CO(2) tensions on oxygenation, pulmonary blood flow (Qp), cerebral blood flow, and systemic blood flow (Qs) after the bidirectional superior cavopulmonary anastomosis (BCPA). BACKGROUND: Hypoxemia refractory to management of a high pulmonary vascular resistance index (PVRI) may complicate recovery from the BCPA. METHODS: After BCPA, CO(2) was added to the inspired gas of mechanically ventilated patients. The Qp, Qs, PVRI, and systemic vascular resistance index (SVRI) were calculated from oxygen consumption, intravascular pressures, and oxygen saturations. Cerebral blood flow was estimated by near infrared spectroscopy and transcranial Doppler. RESULTS: In nine patients (median age 7.1, range 2 to 23 months), arterial oxygen tension increased significantly (p < 0.005) from 36 +/- 6 mm Hg to 44 +/- 6 to 50 +/- 7 mm Hg at arterial carbon dioxide tensions (PaCO(2)) of 35, 45, and 55 mm Hg, respectively and decreased to 40 +/- 8 mm Hg at PaCO(2) 40 mm Hg. At a PaCO(2) of 55 and 45 compared with 35 mm Hg, Qp, cerebral blood flow, and Qs increased significantly, PVRI, Qp/Qs, and the ratio of Qp to inferior vena caval blood flow were unchanged, but SVRI decreased. CONCLUSIONS: We have demonstrated that after the BCPA, systemic oxygenation, Qp, Qs, and cerebral blood flow increased and SVRI decreased at CO(2) tensions of 45 and 55 mm Hg compared with 35 mm Hg. We suggest that hypoxemia after the BCPA is ameliorated by a higher PaCO(2) and that low PaCO(2) or alkalosis may be detrimental. Hypercarbic management strategies may allow earlier progression to the BCPA, which may contribute to reducing the interval morbidity in patients with a functional single ventricle.     

Anatomy of the pulmonary veins in patients with atrial fibrillation and effects of segmental ostial ablation analyzed by computed tomography

INTRODUCTION: The anatomic arrangement of pulmonary veins (PVs) is variable. No prior studies have quantitatively analyzed the effects of segmental ostial ablation on the PVs. The aim of this study was to determine the effect of segmental ostial radiofrequency ablation on PV anatomy in patients with atrial fibrillation (AF). METHODS AND RESULTS: Three-dimensional models of the PVs were constructed from computed tomographic (CT) scans in 58 patients with AF undergoing segmental ostial ablation to isolate the PVs and in 10 control subjects without a history of AF. CT scans were repeated approximately 4 months later. PV and left atrial dimensions were measured with digital calipers. Four separate PV ostia were present in 47 subjects; 3 ostia were present in 2 subjects; and 5 ostia were present in 9 subjects. The superior PVs had a larger ostium than the inferior PVs. Patients with AF had a larger left atrial area between the PV ostia and larger ostial diameters than the controls. Segmental ostial ablation resulted in a 1.5 +/- 3.2 mm narrowing of the ostial diameter. A 28% to 61% focal stenosis was present 7.6 +/- 2.2 mm from the ostium in 3% of 128 isolated PVs. There were no instances of symptomatic PV stenosis during a mean follow-up of 245 +/- 105 days. CONCLUSION: CT of the PVs allows identification of anatomic variants prior to catheter ablation procedures. Segmental ostial ablation results in a significant but small reduction in ostial diameter. Focal stenosis occurs infrequently and is attributable to delivery of radiofrequency energy within the PV.     

Endogenous endothelin-1 limits exercise-induced vasodilation in hypertensive humans

Essential hypertension is a common disorder, associated with increased endothelin-1-mediated vasoconstrictor tone at rest. We hypothesized that increased vasoconstrictor activity of endothelin-1 might explain why the normal decrease in peripheral vascular resistance in response to exercise is attenuated in hypertensive patients. Therefore, we investigated the effect of endothelin A (ET(A)) receptor blockade on the vasodilator response to handgrip exercise. Forearm blood flow responses to handgrip exercise (15%, 30%, and 45% of maximum voluntary contraction) were assessed in hypertensive patients and matched normotensive subjects, before and after intra-arterial infusions of the ET(A) receptor antagonist BQ-123; a control dilator, hydralazine; and placebo (saline). Preinfusion (baseline) vasodilation in response to exercise was significantly attenuated at each workload in hypertensive patients compared with normotensive subjects. Intra-arterial infusions of hydralazine and saline did not increase the vasodilator response to exercise in either hypertensives or normotensives at any workload. The vasodilator response to exercise was markedly enhanced after BQ-123 at the 2 higher workloads in hypertensives (157+/-48%, P<0.01; 203+/-58%, P<0.01) but not in normotensives. This suggests that the impaired vasodilator response to exercise in hypertensive patients is, at least in part, a functional limitation caused by endogenous ET(A) receptor-mediated vasoconstriction. Treatment with endothelin receptor antagonists may, therefore, increase exercise capacity in essential hypertension.