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Telomerase is a specialized ribonucleoprotein polymerase that directs the synthesis of telomere repeats at chromosome ends. Accumulating evidence has indicated that telomerase is stringently repressed in normal human somatic tissues but reactivated in cancers and immortal cells, suggesting that reactivation of telomerase plays an important role in carcinogenesis. In this study, the status of telomerase activity in diseased human nasopharyngeal lesions was determined by the telomeric repeat amplification protocol (TRAP). Fifty-four patients participated including 17 inflammation or hyperplasia, eight with squamous metaplasia, and 29 with different stages of carcinomas. Telomerase activity was detected in 1 of 17 (5.9%) inflammatory or lymphoid hyperplastic tissues, 3 of 8 (37.5%) squamous metaplastic, and 25 of 29 (86.2%) carcinoma tissues. The differences in telomerase expression in these groups is statistically significant (P < 0.001). Levels of telomerase activity correlated with tumour stage (P = 0.024). These results suggest that telomerase reactivation plays a role in the carcinogenesis of nasopharyngeal cancer. Since telomerase activity is found in the majority of nasopharyngeal cancers and a subset of metaplasia, this enzyme may be served as a reference to monitoring the status of abnormal nasopharyngeal tissues. [ABSTRACT FROM AUTHOR]

Background: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen., Objective: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion., Methods: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed., Results: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen., Conclusions: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.

Objectives: Hemodynamic derangement during displacement of beating heart in off-pump coronary artery bypass graft (OPCAB) surgery might be related with right ventricular (RV) dysfunction. We evaluated RV function and hemodynamic alterations using a thermodilution pulmonary artery catheter., Methods: The study included 30 patients undergoing OPCAB, using single pericardial suture and tissue stabilizer. A thermodilution pulmonary artery catheter for continuous monitoring of the cardiac output (CO), right ventricular ejection fraction (RVEF) and RV volume was inserted before anesthesia. The hemodynamic variables were measured after the induction of anesthesia, 5 min after the heart was positioned for each coronary anastomosis and after the sternum was closed., Results: There was no significant change in the RVEF and cardiac index during anastomosis of the left anterior descending artery and right coronary artery. However, the significantly reduced RVEF accompanied by an increase in RV afterload and decrease in the CO was observed during anastomosis of the obtuse marginal (OM) artery. RV volumes did not significantly change during anastomoses, though the right atrial pressure increased during anastomoses of all coronary arteries., Conclusions: The displacement of beating heart for positioning during anastomosis of the graft to OM artery caused significant derangement of RV function and decrease in CO. A thermodilution catheter continuously measuring the CO and RVEF was useful to monitor the change in RV function and volume during OPCAB.

In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.

Extracranial carotid artery dissection may manifest as arterial stenosis or occlusion, or as dissecting aneurysm formation. Anticoagulation and/or antiplatelet therapy is the first-line treatment, but because it is effective and less invasive than other procedures, endovascular treatment of carotid artery dissection has recently attracted interest. We encountered two consecutive cases of trauma-related extracranial internal carotid artery dissection, one in the suprabulbar portion and one in the subpetrosal portion. We managed the patient with suprabulbar dissection using a self-expandable metallic stent and managed the patient with subpetrosal dissection using a balloon-expandable metallic stent. In both patients the dissecting aneurysm disappeared, and at follow-up improved luminal patency was observed.

In 1998, an outbreak of enterovirus 71-associated hand, foot, and mouth disease occurred in Taiwan. Pathologic studies of two fatal cases with similar clinical features revealed two different causative agents, emphasizing the need for postmortem examinations and modern pathologic techniques in an outbreak investigation.

We report a fatal case of enterovirus type 71 (EV 71) infection in an 8-year-old girl during a summer outbreak of hand, foot, and mouth disease in 1998 in Taiwan. The clinical course was rapidly progressive, with manifestations of hand, foot, and mouth disease, aseptic meningitis, encephalomyelitis, and pulmonary edema. The patient died 24 hours after admission. Postmortem study revealed extensive inflammation in the meninges and central nervous system and marked pulmonary edema with focal hemorrhage. Brain stem and spinal cord were most severely involved. The inflammatory infiltrates consisted largely of neutrophils involving primarily the gray matter with perivascular lymphocytic cuffing, and neuronophagia. The lungs and heart showed no evidence of inflammation. EV 71 was isolated from the fresh brain tissues and identified by immunofluorescence method with type-specific EV 71 monoclonal antibody. It was also confirmed by neutralization test and reverse-transcriptase polymerase chain reaction with sequence analysis. The present case was the first example in which EV 71 was demonstrated to be the causative agent of fatal encephalomyelitis during its epidemic in Taiwan.

Sjögren's syndrome is a chronic autoimmune disorder characterized by lymphocytic infiltration of the lacrimal and salivary glands, leading to dryness of eyes (kerato-conjunctivitis sicca) and mouth (xerostomia). The skin lesions in Sjögren's syndrome are usually manifested as xeroderma, but sometimes appear as annular erythema or vasculitis. Central nervous system symptoms may be presented as one of extraglandular manifestations, though rare in incidence, and need differential diagnosis from multiple sclerosis. We report a case of a 45-year-old woman diagnosed as multiple sclerosis at first but later as neurologic manifestation of primary Sjögren's syndrome, showing signs of multiple sclerosis and cutaneous erythematous lesions.

Effort to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimeric gag-env virus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2 gag precursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1IIIB, HIV-1MN, HIV-1RF, and HIV-1SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purified gag-env VLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than the gag VLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1IIIB, HIV-1MN, HIV-1RF, and the gp120 antiserum against HIV-1SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2 gag can be used as a potential HIV vaccine against various HIVs.

The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the "flow adhesion assay") for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.

Platelet adhesion to the exposed surface of the extracellular matrix in flowing blood is the first and critical reaction for in vivo thrombus formation. However, the mechanism of this in vivo platelet adhesion has yet to be studied extensively. One of the reasons for this is the lack of a practical assay method for assessing platelet adhesion under flow conditions. We have devised an assay method (the fluorescent adhesion assay) that is based on the technique originally reported by Hubbell and McIntire (Biomaterials 7:354, 1986) with some modifications to make it more amenable for assaying small samples and have developed an analysis method to quantify the extent of platelet adhesion and aggregation from fluorescence images by using a computer-assisted image analysis system. In our assay, platelet adhesion, expressed as the percentage of the area covered by adhered platelets, was found to increase biphasically as a function of time. In the first phase, platelets interacted with the coated collagen, transiently stopping on the surface; we called this reaction the temporary arrest. In the second phase, platelets adhered much more rapidly and permanently on the surface, and this adhesion was dependent on the shear rate; platelets formed aggregates in this phase. We used our assay to analyze the effects of platelet aggregation inhibitors on platelet adhesion. All three examined inhibitors, EDTA (10 mmol/L), antiglycoprotein (GP) IIb/IIIa, and GRGDS peptide (1 mmol/L), inhibited the second phase adhesion in flowing blood. Furthermore, GPVI-deficient platelets also showed defective second-phase adhesion under the same conditions. These results suggested that GPIIb/IIIa activation and GPVI contribute to the reaction inducing the second phase. The second-phase adhesion has been extensively investigated, and the consensus is that this reaction is mainly attributable to the platelet-platelet interaction. In this report, we were able to detect an earlier reaction, the temporary arrest. This temporary arrest would reflect the fast and weak interaction between platelet GPIb/IX and collagen-von Willebrand factor complexes on the collagen-coated surface.

The genetic basis for Glanzmann's thrombasthenia (GT) was elucidated on a compound heterozygote with glycoprotein (GP)IIb gene: an opal mutation at the end of exon 17 (CGA----TGA) results in only a trace amount of GPIIb mRNA, and a splicing mutation at the acceptor site of exon 26 (CAG----GAG) causes an in-frame, exon skipping process from exon 25 to 27. This aberrant transcript encodes a single-chain polypeptide characterized by a 42-amino acid deletion, which includes the proteolytic cleavage site(s) and a unique, proline-rich region at the location corresponding to the carboxyl-terminal of the normal GPIIb alpha-chain. These characteristics are shared by a previously reported defective GPIIb molecule, which is neither assembled with GPIIIa nor transported to the cellular surface. Despite its normal transcription level, expression of the present defective GPIIb molecule was significantly decreased (approximately 6% of the control level). Because the precursor GPIIb molecule is assembled with GPIIIa in the endoplasmic reticulum (ER) and its processing, as well as stability, is dependent on the GPIIIa subunit, the defective GPIIb molecule may be rapidly degraded by the intrinsic quality control system of the ER due to its inability to form a stable heterodimer complex as a consequence of its misfolded structure. Although we did not confirm that the GPIIIa genes of this individual were normal, GPIIIa may be secondarily decreased (approximately 11% of control), because a large part of it could not be complexed, making it vulnerable to proteolysis. To elucidate the molecular basis for GT, we propose here a classification of GT based on the biosynthetic pathway of the GPIIb-IIIa complex.

Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.

We describe an individual with abnormal platelet glycoprotein (GP) IIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IIIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.