Site-directed mutagenesis of coumarin-type anticoagulant-sensitive VKORC1: evidence that highly conserved amino acids define structural requirements for enzymatic activity and inhibition by warfarin

Coumarin and homologous compounds are the most widely used anticoagulant drugs worldwide. They function as antagonists of vitamin K, an essential cofactor for the posttranslational gamma-glutamyl carboxylation of the so-called vitamin K-dependent proteins. As vitamin K hydroquinone is converted to vitamin K epoxide (VKO) in every carboxylation step, the epoxide has to be recycled to the reduced form by the vitamin K epoxide reductase complex (VKOR). Recently, a single coumarin-sensitive protein of the putative VKOR enzyme complex was identified in humans (vitamin K epoxide reductase complex subunit 1, VKORC1). Mutations in VKORC1 result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2). Here,we report on the expression of site-directed VKORC1 mutants, addressing possible structural and functional roles of all seven cysteine residues (Cys16, Cys43, Cys51, Cys85, Cys96, Cys132, Cys135), the highly conserved residue Ser/Thr57, and Arg98, known to cause VKCFD2 in humans. Our results support the hypothesis that the C132-X-X-C135 motif in VKORC1 comprises part of the redox active site that catalyzes VKO reduction and also suggest a crucial role for the hydrophobic Thr-Tyr-Ala motif in coumarin binding. Furthermore, our results support the concept that different structural components of VKORC1 define the binding sites for vitamin K epoxide and coumarin.     

Cyclooxygenase-2 inhibitors suppress the growth of human hepatocellular carcinoma implants in nude mice

Cyclooxygenase (COX)-2 is expressed in hepatocellular carcinomas (HCCs) and HCC cell lines. COX-2 inhibition strongly suppresses growth of HCC cells in vitro by inducing apoptosis and reducing proliferation. Here, we evaluate the in vivo effects and mechanism of COX-2 inhibition of human HCC cell line derived xenotransplanted tumors in nude mice. Firstly, nude mice were treated with a COX-2 specific inhibitor (meloxicam) or a non-specific inhibitor (sulindac) starting 5 days prior to tumor cell injection. After 35 days mice were killed and tumors were analyzed morphologically and assayed for proliferation (Ki67), apoptosis (M30) and COX-2 expression. Secondly, mice were treated with meloxicam or sulindac after tumors had reached a diameter of at least 0.2 cm. COX-2 expression was maintained in implant tumors at levels comparable with parental cells. Selective COX-2 inhibition led to a significant reduction of tumor growth and weight. COX-2 inhibition had a significant anti-proliferative and pro-apoptotic effect on tumor cells. These results demonstrate that under experimental conditions selective COX-2 inhibition suppresses solid HCC growth in vivo and, therefore may have preventive and therapeutic potential for human HCCs.     

Bone marrow microenvironment facilitating dendritic cell: CD4 T cell interactions and maintenance of CD4 memory

This study shows that bone marrow (BM) stroma expresses constitutively multiple adhesion molecules (ICAM-1, VCAM-1, MadCAM-1, P-selectin) relevant for the homing and infiltration of BM by blood derived T lymphocytes, and also the co-stimulatory molecule CD80, relevant for T cell activation. T cells were capable of homing to BM but not to thymus. Homing to BM involved the integrins LFA-1alpha and alpha4 which interact with the above constitutively expressed cell adhesion molecules (CAMs). CD3 T cells were detected together with BM resident CD11c dendritic cells (DCs), often enriched in follicle-like structures in BM parenchyma. Cognate interactions between transferred antigen specific transgenic CD4 T cells and antigen laden BM-DCs led to formation of multicellular clusters in situ in BM, to generation of lymphoblasts and to clonal T cell expansion within such clusters. The great majority of BM-CD4 T cells had a memory phenotype suggesting that the BM microenvironment facilitates maintenance of CD4 memory. These results extend and corroborate our previous findings on BM-CD8 T cell mediated immune responses. Together these findings suggest that DC-T cell interactions in BM play an important role in immune responses to blood-borne antigen and in the establishment of systemic immunity and long-term memory.     

Infections preceding early arthritis in southern Sweden: a prospective population-based study

OBJECTIVE: To detect evidence of infections preceding early arthritis in Southern Sweden and to compare the clinical outcome of remission during a 6-month followup for patients with and without signs of prior infection. METHODS: Adult patients with arthritis of less than 3 months' duration were referred from primary health care centers to rheumatologists. All patients were systematically screened for infections caused by Salmonella typhimurium and Salmonella enteritidis, Yersinia enterocolitica, Campylobacter jejuni, Borrelia burgdorferi, Chlamydia trachomatis, Chlamydia pneumoniae, and parvovirus B19. RESULTS: Seventy-one patients were included in this study. Twenty-seven (38%) patients had reactive arthritis (ReA), 17 (24%) undifferentiated arthritis, 15 (21%) rheumatoid arthritis (RA), 4 (6%) psoriatic arthritis, and the rest (11%) other diagnoses. Of all the patients, 45% had evidence of a recent infection preceding the arthritis, as indicated by laboratory tests and/or disease history. C. jejuni dominated the ReA group. The occurrence of recent C. trachomatis, B. burgdorferi, C. pneumoniae, and parvovirus B19 infections was low. Overall, 58% of the patients went into remission during the 6-month followup. Of the patients with a preceding infection, 69% went into remission as compared to 38% of the patients without a preceding infection (p = 0.011). Thirty-three percent of the patients with RA were in remission after 6 months. CONCLUSION: In this population-based cohort, 45% of the patients presenting with a new-onset arthritis had had a prior infection. Campylobacter ReA dominated the ReA group. There were only a few cases preceded by infections by C. trachomatis, B. burgdorferi, C. pneumoniae, and parvovirus B19 infections. Remission during the first 6 months was especially frequent in the group of patients with a prior infection, but the remission rate was relatively high even for arthritis without prior infection.     

18q-syndrome: brain MRI shows poor differentiation of gray and white matter on T2-weighted images

PURPOSE: To study brain MRI findings in patients with 18q- syndrome and to correlate these findings with the results of the molecular breakpoint analysis. MATERIALS AND METHODS: Brain MR images of 17 patients with 18q- syndrome were evaluated. Segregation analysis was performed with 15 microsatellite markers to determine the deletion breakpoints and whether the deletion included the myelin basic protein (MBP) gene. RESULTS: One patient had an interstitial deletion of 18q which spared the MBP gene. He was the only one with normal brain MRI. All 16 patients with deletions including the MBP gene had abnormal white matter in MRI. The main finding was poor differentiation of gray and white matter on T2-weighted images due to increased white matter signal intensity. In addition, measured signal intensity of the white matter was significantly increased in patients compared with controls. CONCLUSIONS: Poor differentiation of gray and white matter on T2-weighted images is the most typical MRI finding of the 18q- syndrome. These results support the postulation that abnormal myelination in 18q- syndrome is due to haploinsufficiency at or near the MBP locus.     

Lowering of LDL cholesterol rather than moderate weight loss improves endothelium-dependent vasodilatation in obese women with previous gestational diabetes

OBJECTIVE: Effects of weight loss on vascular function are unknown. We compared, in the face of similar weight loss over 3-6 months, effects of orlistat (120 mg t.i.d., n = 23) and placebo (n = 24) on in vivo endothelial function in a high-risk group of obese (BMI 32.1 +/- 0.4 kg/m(2)) premenopausal nondiabetic women with a history of gestational diabetes. RESEARCH DESIGN AND METHODS: Forearm blood flow responses to intra-arterial infusions of acetylcholine (ACh) and sodium nitroprusside (SNP), body composition, and serum lipids were determined before and after weight loss. RESULTS: Weight loss averaged 7.3 +/- 0.2 kg (8.3 +/- 0.1%) and 7.4 +/- 0.2 kg (8.2 +/- 0.1%) of initial body weight in the orlistat and placebo groups, respectively. Forearm and body compositions changed similarly in both groups. Responses to ACh increased by 41% to the low dose (5.9 +/- 0.6 vs. 8.3 +/- 0.3 for flow in the experimental/control arm, P < 0.01) and by 33% to the high dose (7.6 +/- 0.8 vs. 10.1 +/- 0.6, P < 0.001) in the orlistat group, but they remained unchanged in the placebo group. The blood flow responses to SNP did not differ significantly between the groups. LDL cholesterol decreased significantly in the orlistat group from 3.5 +/- 0.2 to 3.0 +/- 0.1 mmol/l (P < 0.01) but remained unchanged in the placebo group. Within the orlistat group, the decrease in LDL cholesterol correlated significantly with the improvement in the blood flow response to ACh (r = -0.44, P < 0.05). CONCLUSIONS: Orlistat but not moderate (8%) weight loss per se improves endothelial function in women with previous gestational diabetes. This improvement is associated with a lowering of LDL cholesterol by orlistat.     

Underlying factors in the association between depressed mood and mobility limitation in older people

BACKGROUND: Depressed mood may either precede mobility limitation or follow from mobility limitation. OBJECTIVE: To compare mood status among people with manifest mobility limitation, those with preclinical mobility limitation and those without mobility limitation and investigate factors explaining the association between depressed mood and mobility limitation. DESIGN: Cross-sectional. Subjects: 645 community-living 75- to 81-year-old people. METHODS: Depressed mood was assessed using the Centre for Epidemiologic Studies Depression Scale (CES-D, cut-off score 16); difficulty walking 500 m was assessed by self-report. Those reporting difficulty were categorised as having manifest mobility limitation. Those with no difficulty but reporting task modifications, such as reduced frequency of walking, were categorised as having preclinical mobility limitation. The association between depressed mood and mobility limitation was analysed using logistic regression analysis with gender, age, economic situation, the availability of a confidant, chronic conditions, and widespread pain as covariates. RESULTS: Depressed mood was found in 34% of subjects with manifest mobility limitation, in 26% of those with preclinical mobility limitation, and in 13% of those without mobility limitation. The unadjusted odds ratio for depressed mood was 3.43 (95% CI 2.04-5.76) among subjects with manifest mobility limitation and 2.38 (95% CI 1.52-3.73) among those with preclinical mobility limitation, compared to those without mobility limitation.Adjustment for covariates reduced the risks to 2.10 (95% CI 1.15-3.82) and 1.99 (95% CI 1.24-3.20), respectively. Widespread pain explained 28% of the increased risk of depressed mood among those with manifest mobility limitation. CONCLUSION: The dose-response relationship between depressed mood and mobility limitation suggests that both conditions may progress simultaneously and may share aetiology, at least in part. Pain may be an underlying factor in both depressed mood and mobility limitation.