Alternative splicing gives rise to two novel long isoforms of Zn-alpha 2-glycoprotein, a member of the immunoglobulin superfamily

The integrin alpha IIb beta 3 (GPIIb/IIIa) mediates platelet aggregation by a change in affinity for the ligand fibrinogen. The amino acids 991-995 (GFFKR) at the NH2-terminus of the cytoplasmic domain are highly conserved in all known integrin alpha subunits. We postulated that the GFFKR-region is important for the inside-out signal transduction and has an influence on the affinity state of integrins. To test this hypothesis, a mutant with a deletion in the GFFKR region was designed. The DNA-constructs were constructed by PCR, sequenced, cotransfected with the beta 3 subunit into CHO cells and cell surface expression was proven with immunoprecipitation and flow cytometry. The GFFKR-deletion mutant demonstrated a high affinity binding of the mAb PAC-1 and I125-labeled fibrinogen. The metabolic inhibitors 2-deoxyglucose and NaN3 did not change the affinity state of the deleted receptor. Neither did the truncation of the cytoplasmic domain of the beta 3 subunit. Additionally, expression of the deleted integrin in the erythropoetic cell line K562 revealed a high affinity state. A deletion of the GFFKR-region in the cytoplasmic domain of the alpha subunit locks integrin alpha IIb beta 3 in a high affinity state. This is an intrinsic property of the deleted receptor since there is no energy dependence and no cell type specifity. Thus, the GFFKR-region is involved in inside-out signaling in alpha IIb beta 3. Furthermore, cell lines expressing this activated alpha IIb beta 3 integrin may be used as models for activated platelets.     

Renal handling of Ca2+ in diabetes

Transient activation of COS-1 cell phospholipase-D (PLD) in response to the protein kinase C (PKC) agonist tetradecanoyl phorbol acetate (TPA) was demonstrated by monitoring the ethanol-dependent accumulation of phosphatidylethanol (PtdEth). Transfection of COS-1 cells with PKC-alpha (wild type and constitutively activated mutants) produced no detectable ptdEth on incubation of transfected cells in the presence of ethanol. However, the response of transfected cells to subsequent TPA stimulation was inhibited, consistent with a role for the PKC-alpha in the suppression of PLD activity.     

Anti-beta 2-glycoprotein I autoantibodies from patients with the "antiphospholipid" syndrome bind to beta 2-glycoprotein I with low affinity: dimerization of beta 2-glycoprotein I induces a significant increase in anti-beta 2-glycoprotein I antibody affinity

"Antiphospholipid" autoantibodies are associated with arterial and venous thrombosis, recurrent fetal loss, and thrombocytopenia. At present, the best-characterized antigenic target for these autoantibodies (or Abs) is the phospholipid-binding protein beta2-glycoprotein I (beta2GPI). These Abs bind beta2GPI only in the presence of negatively charged phospholipids or microtiter polystyrene plates that have been specially treated to give the surface a negative charge. To determine whether the binding of these Abs to beta2GPI on negatively charged surfaces is dependent on increased density or neo-epitopes formed as a consequence of a conformational change on beta2GPI, we generated mutants of beta2GPI by site-directed mutagenesis and assessed the binding characteristics of anti-beta2GPI Abs to these mutants. Our results demonstrate that mutant F307*, which spontaneously forms significant dimerization, is bound best by all the anti-beta2GPI Abs in an anti-beta2GPI ELISA using irradiated polystyrene microtiter plates. In addition, these Abs bound mutant F307* coated onto standard polystyrene microtiter wells in the absence of phospholipid, whereas there was minimal binding with wild-type and mutant F307*/C288A, which formed minimal dimerization. Affinity-purified anti-beta2GPI Abs from patients with the antiphospholipid syndrome demonstrated significantly higher binding affinity for mutant F307* in fluid phase than for wild-type or mutant F307*/C288A of beta2GPI. These results demonstrate that autoantibody binding to beta2GPI is intrinsically of low affinity and that the binding is dependent on the density of the Ag and not on neo-epitope formation.     

Cisplatin-DNA adduct determination in the hepatic albumin gene as compared to whole genomic DNA

A quantitative time-resolved fluorometriC PCR-stop assay has been used to determine cisplatin-DNA adducts in a 1.612 kb region and a polymorphic 1.85 kb region of the rat liver albumin gene containing parts of exons B and C and all of the BC intron. The values were compared to adducts in the whole rat liver genome determined by atomic absorbance spectrometry (AAS). Initial validation of the PCR-stop assay involved modification of purified rat liver DNA in vitro to desired levels by incubation with different concentrations of cisplatin. In these DNA samples, cisplatin-DNA adduct levels determined in the 1612 base pair fragment by PCR-stop assay were shown to be similar to those determined in the whole genomic DNA by AAS. In freshly isolated primary rat hepatocytes cultured for 2 h with 50, 75, 100, and 150 microM cisplatin, adduct levels determined by the PCR-stop assay were similar to those measured by AAS. Cultured MH1C1 rat hepatoma cells, which express albumin, had a polymorphism in the rat albumin gene such that the fragment amplified with the same primers was about 1.85 kb (13% larger). When MH1C1 cells were exposed to 5, 15, 25, 50, and 75 microM cisplatin for 24 h, 50% cell kill was at 21.0 +/- 5.5 microM cisplatin. For doses of 15-75 microM cisplatin, the cisplatin-DNA adduct levels in this fragment, measured by PCR-stop assay, were about one-half of those in the whole genomic DNA measured by AAS. In addition, MH1C1 cells exposed to 150 microM cisplatin for 4 h and subsequently incubated with fresh medium for 24 h showed no change in adduct level in whole genomic DNA during this time but showed a 29% adduct removal in the 1.85 kb fragment. The data demonstrate that this 1.85 kb region containing expressed regions of the albumin gene has undergone both less adduction and more rapid adduct removal, as compared to the MH1C1 genome as a whole.     

Regulation of Zn-alpha2-glycoprotein-mediated cell adhesion by kininogens and their derivatives

MC3T3-E1 (mouse osteoblast-like) cells adhered to a tissue culture plate coated with human Zn-alpha2-glycoprotein (Znalpha2gp). The adhesion of MC3T3-E1 cells to Znalpha2gp was inhibited by synthetic peptides such as RGDS and ELRGDV, and by antibody against vitronectin receptor. These findings suggested that the RGD region of Znalpha2gp interacts with the vitronectin receptor (alphavbeta3) on the MC3T3-E1 cell surface. Furthermore, we found that the common heavy chain of both HMW- and LMW-kininogens accelerated the Znalpha2gp-mediated MC3T3-E1 cell adhesion. Among the three domains of the common heavy chain of both kininogens, domain 3 promoted the cell adhesion by up to 200%. Among the nine synthetic peptides covering domain 3, the peptide, N334AEVYVVPWEKKIYPTVN351 accelerated in a dose-dependent manner the Znalpha2gp- and vitronectin (VN)-mediated MC3T3-E1 cell adhesion. These findings suggested that a defined region of domain 3 is responsible for the acceleration of cell adhesion.     

Renal function in cystic fibrosis with special reference to the renal sodium handling

Renal function was studied in 16 patients with cystic fibrosis, aged 5 to 19 years. The mean glomerular filtration rate and filtration fraction were increased compared to those in controls. Basal urinary sodium excretion as well as renal responses to oral and intravenous sodium loads were low, indicating an increased renal sodium reabsorption. Diluting capacity, measured as free water clearance, was decreased because of a low distal sodium delivery which might indicate an increased proximal sodium reabsorption.     

Differential effects of anti-beta2-glycoprotein I and antiprothrombin antibodies on the anticoagulant activity of activated protein C

Antiprothrombin and anti-beta2-glycoprotein I (beta2-GPI) antibodies belong to the family of antiphospholipid (APL) antibodies and represent the phospholipid-dependent inhibitors of coagulation. They may be distinguished by analyzing the coagulation profiles generated by the comparison of the ratios of two coagulation tests, the Kaolin Clotting Time (KCT) and the dilute Russell's Viper Venom Time (dRVVT), commonly adopted for their diagnosis. The KCT profile is caused by antiprothrombin antibodies, whereas anti-beta2-GPI antibodies are responsible for the dRVVT coagulation profile. The presence of aPL antibodies is frequently associated with acquired resistance to activated Protein C (APC-R), but limited information is available regarding the role of the different antibodies in its development. We studied the time-course of activated Factor V (FVa) generation and inactivation in the plasma of 42 patients with well-defined phospholipid-dependent inhibitors of coagulation: 24 displayed the dRVVT coagulation profile, whereas the other 18 cases showed the KCT profile. In normal pooled plasma, the peak values of FVa (mean +/- standard deviation, [SD]: 16.307 +/- 4.372 U/mL) were reached in 4 to 5 minutes and an almost complete inactivation (0.088 +/- 0.123 U/mL) was obtained within 20 minutes. At this time point, values of residual FVa exceeding 2 SD the mean of controls (0.344 U/mL) were considered abnormal. Patients belonging to the KCT coagulation profile group reached the maximal amount of FVa in plasma (22.740 +/- 7.693 U/mL, P = not significant v controls) within 4 to 5 minutes; at 20 minutes, the residual amount of FVa in plasma ranged from 0 to 1.09 U/mL (0.293 +/- 0.298; P = .027), but it was found abnormal in only six of the 18 cases. The time-course of FVa in plasma of patients belonging to the dRVVT coagulation profile group differed from that of normal controls in that the peak values (10.955 +/- 5.092 U/mL) were reached at 10 minutes and the amount of residual FVa at 20 minutes ranged from 0.320 to 14.450 U/ml (2.544 +/- 3.580 U/mL; P = .0191 v normal controls and P = . 0114 v KCT group patients). Twenty of the 24 patients belonging to the dRVVT profile group had an abnormal inactivation of FVa (chi2 = 0.001 v KCT group patients). History of venous thrombosis was experienced by 15 patients: an abnormal rate of FVa inactivation was found in 11 of them (73%) versus 15 of the 27 cases without thrombosis (56%) (x2 = 0.2556). The effect of affinity-purified IgG phospholipid-dependent inhibitors of coagulation on the time-course of FVa generation and inactivation in normal plasma was also investigated. Anti-beta2-GPI, but not antiprothrombin antibodies, hampered the inactivation of FVa by endogenous APC, thus reproducing the behavior of the original plasmas. This effect was strictly beta2-GPI-dependent. In conclusion, our findings confirm that anti-beta2-GPI antibodies identify patients with phospholipid-dependent inhibitors of coagulation at increased risk of thrombosis and suggest acquired APC-R as a possible explanation of the pathogenesis of the thromboembolic events.     

The cost of dental implants as compared to that of conventional strategies

The effectiveness of dental implants is widely studied, especially in terms of their clinical outcomes. However, from the policymaker's point of view, variables other than safety and efficacy, such as the costs and effectiveness of dental implants as compared to other treatment alternatives, are vital in decision making. This paper compares the costs of different treatment strategies in a randomized clinical trial in patients with resorbed mandibles and persistent problems with their conventional dentures: treatment with a mandibular overdenture on permucosal dental implants, an overdenture on a transmandibular implant, new dentures after preprosthetic surgery, and new dentures only. Data were gathered on an individual patient level to gain insight into specific cost episodes. Direct costs were subdivided into labor, material, technique, and overhead. Data concerning these components were gathered during the consecutive treatment phases in the first year. Results show that the resources used to treat a patient with an overdenture supported by a transmandibular implant are seven times those of a complete new set of dentures. Comparison of the cost ratio of an implant-retained overdenture supported by permucosal implants and conventional new prostheses proves less unfavorable: 1:3. New dentures after preprosthetic surgery are almost as expensive as treatment with permucosal implants.     

Renal handling of carnitine in ill preterm and term neonates

BACKGROUND/AIMS: A prospective study was performed to evaluate the incidence of bile leak after T-tube removal, using 99mTc labelled BULIDA scintigraphy. PATIENTS AND METHODS: Thirty seven patients with choledocholithiasis underwent choledocholithotomy and T-tube drainage. T-tubes were removed on 9--26 postoperative day (mean 13.4 days). RESULTS: Six (16%) patients had scintigraphic evidence of bile leak; 4 patients had leak demonstrated in the early phase only. One patient each had partial persistent leak and complete bile leak. All patients were managed nonsurgically; conservative treatment: (4), percutaneous drainage (PCD) of intra-abdominal collection (1) and endoscopic sphincterotomy and PCD of intra-abdominal collection (1). There was no significant difference in the leak rate when T-tubes were removed at < 2 weeks versus later than 2 weeks (P=.97). CONCLUSIONS: T-tubes should be removed with extreme caution and treatment of symptomatic patients should be guided by clinical and scintigraphic findings.     

Variability in the arterial pressure of newborns as compared to that of adults and old men

The systolic and diastolic blood pressure was monitored on healthy subjects of different ages, spanning from newborns (24-48 hs old, to elderly age (70-80 years old). Each subject was monitored for 15 minutes, and the pressure values collected every minute. This protocol allowed a statistical evaluation of both inter- and intraindividual variabilities among and within different class ages. As expected, increasing age is paralleled by a marked increase in the average values of systolic blood pressure; the trend of variabilities around the average, however, as traced by standard deviations, is just the opposite: a marked decrease going from newborns to adults is accompanied--in most cases--by a statistically significant difference in the same direction also between adults and old men. This can be rationalized, in the light of recent findings on the role of deterministic chaos in the functional behaviour of complex biological systems, in terms of decreased functional flexibility characteristic of the aging process.     

MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2

The process of post-replicative DNA-mismatch repair seems to be highly evolutionarily conserved. In Escherichia coli, DNA mismatches are recognized by the MutS protein. Homologues of the E. coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals. Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) and human hMSH2 proteins have been shown to bind to mismatch-containing DNA in vitro. However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutS alpha isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP. It has been reported that S. cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2. We show here that, as in human cells, the S. cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP.     

Changes in equine endometrial retinol-binding protein RNA during the estrous cycle and early pregnancy and with exogenous steroids

A cDNA library was constructed from poly(A) RNA obtained from Day 14 nonbred equine endometrium. A cDNA probe for porcine retinol-binding protein (RBP) was used to screen the library, and a complete cDNA sequence (1133 bp, excluding the poly(A) tail) was obtained. Endometrial biopsies were obtained from cycling, nonbred mares at Days 0, 1, 4, 8, 10, 11, 13, and 15 and from pregnant mares at Days 11, 13, 15, and 17 after ovulation (n = 2 mares each day). Endometrial biopsies were also taken from 18 noncycling anestrous mares after the following treatments: C (vehicle control for 1 day, n = 3), E (estradiol-17 valerate, 5 mg/day for 6 days, n = 3), P (progesterone, 250 mg/day for 6 days, n = 4), ESP (E for 6 days followed by, P for 6 days, n = 4), and ELP (E for 6 days followed by P for 12 days, n = 4). Northern blot analyses were performed on total RNA (30 micrograms) using cDNA probes to equine (e) RBP and human glyceraldehyde-3 phosphate dehydrogenase (G3PDH). The RBP RNA levels (normalized to G3PDH) from nonbred mares were low during early diestrus and increased after Day 10, and RBP RNA levels from pregnant mares were similar to those of nonbred mares for corresponding days. E tended to decrease endometrial RBP RNA; and P, ESP, and ELP increased it compared to C. There were no significant differences among P, ESP, and ELP RBP RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-cardiolipin and anti-beta2-glycoprotein I antibodies in patients with inflammatory bowel disease

Patients with inflammatory bowel disease (IBD) frequently suffer from thromboembolic events. Anti-cardiolipin (aCL) antibodies have been shown to be associated with thrombosis. Recently, the antibodies against the anti-cardiolipin cofactor beta2-glycoprotein I (a(beta2)GPI) have been found with higher specificity for thrombosis. The presence of these antibodies was assessed in 128 patients with IBD [83 with ulcerative colitis (UC) and 45 with Crohn's disease (CD)] and 100 healthy controls (blood donors). Patients with UC and CD had a significantly higher prevalence of aCL (18.1% and 15.6%, respectively) than healthy controls (HC) (3%). Eleven IBD patients (8.6%) but no HC had a(beta2)GPI. None of the IBD patients with a history of thrombosis had aCL and only one of them (a UC patient with deep vein thrombosis of the right leg) had a high titer of IgG a(beta2)GPI. In conclusion, these data show that both aCL and a(beta2)GPI are significantly associated with IBD but further studies are needed to determine the significance of our findings.     

Induction of early atherosclerosis in LDL-receptor-deficient mice immunized with beta2-glycoprotein I

BACKGROUND: Immunization with beta2-glycoprotein I (beta2GPI), the probable target of autoimmune anticardiolipin antibodies, results in experimental antiphospholipid syndrome in different mouse strains. The present study was undertaken to evaluate the effect of beta2GPI immunization on the progression of atherosclerosis. METHODS AND RESULTS: In the first experiment, 3 groups of LDL receptor-deficient (LDL-RD) mice (n=15 per group) were immunized with either beta2GPI or ovalbumin or were not immunized and were fed a chow diet for 12 weeks. In a second experiment, 3 groups of LDL-RD mice (n=10 per group) were immunized similarly and fed an atherogenic diet for 6 weeks. All beta2GPI-immunized mice developed high titers of anti-beta2GPI antibodies as well as a specific lymph node proliferation to beta2GPI. The average cholesterol levels did not differ between the mice fed similar diets, regardless of the immunization protocol. Atherosclerosis was enhanced in the beta2GPI-immunized mice (mean aortic lesion, 26 000+/-5700 microm2) in comparison with their ovalbumin-immunized (mean, 3000+/-1099 microm2; P<0.01) and nonimmunized (mean, 2250+/-700 microm2; P<0.01) littermates. The average lesion size in the beta2GPI-immunized mice fed an atherogenic diet (mean, 98 000+/-8305 microm2) was larger than the ovalbumin-immunized mice (mean, 81 250+/-12 933 microm2; P=NS) or the nonimmunized controls (mean, 75 625+/-7281 microm2; P=NS). The atherosclerotic plaques in the beta2GPI-immunized mice appeared to be more mature, and denser infiltration of CD4 lymphocytes was present in the subendothelium of the aortic sinuses from this group of mice. CONCLUSIONS: The results of the present study provide the first direct evidence for the proatherogenic effect of ss2GPI immunization and establish a new model for immune-mediated atherosclerosis.     

Role of the N- and C-terminal domains of bovine beta 2-glycoprotein I in its interaction with cardiolipin

beta 2-Glycoprotein I (beta 2-GPI) is a cofactor in the recognition of the phospholipid antigen cardiolipin by anti-cardiolipin antibodies in autoimmune diseases such as systemic lupus erythematosus. We examined the interactions of various forms of bovine beta 2-GPI, such as its intact form, desialylated form (Asialo-beta 2-GPI), N-terminal domain (Domain I), and modified forms of beta 2-GPI and Asialo-beta 2-GPI with nicks in their C-terminal domains, with phospholipid liposomes under different conditions of pH and ionic strength. We found that at neutral pH and low ionic strength, beta 2-GPI became bound to liposome membranes containing cardiolipin, phosphatidylglycerol, phosphatidylserine, phosphatidylserine, phosphatidic acid, or phosphatidylinositol, but not phosphatidylcholine alone. The number of phospholipids involved in the binding seemed to depend on the head group structure of the negatively charged phospholipids, but the dissociation constant did not, being about 10(-8) M, except that for the interaction with phosphatidylinositol, which was one order of magnitude lower. We also found that Domain I and Asialo-beta 2-GPI bound to liposome membranes containing negatively charged phospholipids, and that in the interaction with cardiolipin, their dissociation constants were about 10(-6) and 10(-8) M, respectively. At neutral pH and both low and high ionic strengths, the affinities of the nicked forms of beta 2-GPI and Asialo-beta 2-GPI for cardiolipin were both lower than those of their intact forms but similar to that of Domain I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to beta 2-glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus

OBJECTIVE: To investigate whether anticardiolipin antibodies (aCL) in patients with systemic lupus erythematosus (SLE) bind to beta 2-glycoprotein I (beta 2GPI), and to search for a relationship between the presence of IgG and/or IgM anti-beta 2GPI antibody and clinical manifestations in SLE patients. METHODS: IgG and IgM anti-beta 2GPI in 308 Japanese SLE patients were measured using phospholipid-independent enzyme immunoassays. Relationships to clinical histories and to various laboratory data were examined. RESULTS: The values of anti-beta 2GPI and aCL, as measured by conventional enzyme immunoassay, showed a strong correlation, but the anti-beta 2GPI assay was more useful in distinguishing beta 2GPI-dependent aCL from beta 2GPI-independent aCL. The presence of IgG anti-beta 2GPI was associated with an increased frequency of a history of thrombosis. Comparisons of various laboratory data suggested that the titer of anti-beta 2GPI may fluctuate with disease activity. CONCLUSION: The results suggest that pathogenic aCL is directed against structurally altered beta 2GPI and that enzyme immunoassay for anti-beta 2GPI may prove useful in evaluating the risk of thrombosis and monitoring the clinical course in patients with SLE.