Thrombin cleavage-independent deposition of fibrinogen in extracellular matrices

Lung epithelial cells (A549) synthesize and secrete fibrinogen (FBG) in vitro when stimulated with interleukin-6 and dexamethasone. This FBG secretion is polarized in the basolateral direction, suggesting that FBG is a component of the extracellular matrix (ECM). Immunofluorescent staining of A549 cells showed a fibrillar pattern of FBG, similar to the staining detected using antibodies against the matrix constituents, collagen type IV and fibronectin (FN). The same pattern of staining was detected using antibodies against fibrinopeptides A and B, as well as with the T2G1 monoclonal antibody against the fibrin-specific epitope, beta15-21. Matrix staining was unaltered in the presence of the thrombin inhibitor, hirudin, or the plasmin inhibitor, aprotinin, consistent with the interpretation that matrix deposition of FBG does not require such enzymatic action. Metabolic labeling studies confirmed that FBG secreted from A549 cells or deposited into the ECM showed no evidence of thrombin or plasmin proteolytic processing or of transglutaminase-mediated covalent cross-linking (gamma-gamma dimers or alpha-polymers). Incubation of either A549 cell-derived or purified plasma FBG with cultures of human foreskin fibroblasts resulted in FBG deposition in the ECM that colocalized with matrix fibrils containing endogenously produced FN and laminin (LN). Binding of FBG to this exogenously produced matrix was unaltered by inhibition of thrombin and plasmin action, yet also exhibited exposure of the fibrin-specific epitope, beta15-21. The majority (approximately 70%) of newly synthesized and secreted FBG is bound to the cell surface as determined by its trypsin-sensitivity. Cell surface-bound FBG is initially deoxycholate-soluble, which, over time, becomes incorporated in the deoxycholate-insoluble ECM in a similar fashion to FN. These data suggest that matrix incorporation requires the binding of secreted FBG to cell-associated matrix assembly sites. However, unlike FN, FBG in the ECM is composed of the dimeric protamer (A alpha/B beta/gamma gamma) and not high molecular weight polymers indicative of fibrin. This study provides evidence that deposition of FBG in both endogenous and exogenously produced matrices results in conformational changes that occur independently of thrombin cleavage. This matrix-bound FBG, on which unique cell-reactive domains are likely exposed, could augment cellular response mechanisms evoked during injury and inflammation.     

Taxanes propagate apoptosis via two cell populations with distinctive cytological and molecular traits

Gross observation at the slaughterhouse determines the primary selection of porcine aortic valves for the manufacture of bioprostheses. This step is critical because only valves with significant abnormalities are rejected. The present study validated this selection process by investigating the pathological characteristics of one series of accepted valves and one series of rejected valves. Macroscopy, x-ray examination, light microscopy, and scanning electron microscopy (SEM) were performed on 5 initially rejected valves, 3 leaflets from 3 other initially rejected valves, and 6 valves that successfully passed this first step in the selection process. Abnormalities were macroscopically visible only on the rejected valves and were described as thick white areas, heavy white striations, thin spots, white plaques, and nodules. Individual variability in the structure of each leaflet was more significant in the rejected valves than in the valves that had passed the first inspection. The leaflets of the rejected valves were also irregularly thick with a lack of consistency in the position and prominence of the different layers. The formation of nodules and the presence of white plaques in the inner fibrosa layer were among the pathological features. The initially accepted valves considered defect free under gross observation continued to display some weaknesses, and not all of the valves selected during the first step of the process were suitable to become bioprostheses. Because the manufacturer carries out further quality control inspections at every step of preparation resulting in additional rejections, it is therefore anticipated that all valves with defects will be rejected. None of the rejected valves were defect free, and rejection was fully justified.     

Increased urinary excretion of 3-hydroxyisovaleric acid and decreased urinary excretion of biotin are sensitive early indicators of decreased biotin status in experimental biotin deficiency

To assess the utility of various indicators of biotin status, marginal biotin deficiency was induced experimentally in normal adults. Ten subjects consumed a diet that contained enough avidin to bind seven times more biotin than that in the diet. Blood and 24-h urine samples were collected before the diet began and twice weekly thereafter for 20 d. The urinary excretion and serum concentration of biotin and its two principal inactive metabolites bisnorbiotin and biotin sulfoxide were determined after HPLC separation with an avidin-binding assay. The urinary concentration of 3-hydroxyisovaleric acid, an indicator of reduced activity of a biotin-dependent enzyme, was quantitated by gas chromatography-mass spectrometry. The urinary excretion of 3-hydroxyisovaleric acid increased significantly (P < 0.0001). For all subjects, the urinary excretion of both biotin and bisnorbiotin decreased significantly (P < 0.0001 for each). In contrast, the mean serum concentration of biotin did not decrease significantly (P = 0.06). These data provide evidence that the urinary excretion of 3-hydroxyisovaleric acid and the urinary excretion of biotin are early and sensitive indicators of biotin deficiency and that the serum concentration of biotin is not.     

Risedronate in the treatment of Paget's disease of bone: an open label, multicenter study

An open-label, multicenter study was conducted to determine the efficacy and safety of oral risedronate (a pyridinyl bisphosphonate) in 162 patients (102 men, 60 postmenopausal women; mean age, 68 years) with moderate to severe Paget's disease of bone (mean serum alkaline phosphatase [ALP] approximately seven times the upper limit of normal). Patients were treated with oral risedronate, 30 mg/day for 84 days, followed by 112 days without treatment. This 196-day cycle was repeated once if serum ALP did not normalize or increased from the nadir value by > or = 25%. At the end of the first and second cycles, the mean percentage decreases for serum ALP were 65.7% and 69.1%, and for urinary hydroxyproline/creatinine 50.4% and 66.9%, respectively. The decreases from baseline in ALP and urinary hydroxyproline/creatinine were significant (p < 0.001). Normalization of serum ALP was observed in 86 patients (53.8%): 53 during the first treatment cycle and 33 during the second. There was a significant proportion of patients reporting a decrease in the pagetic bone pain at days 84 and 196 (p < 0.001). Overall, risedronate was well tolerated. Five patients withdrew due to adverse events, none of which were considered to be drug related. In conclusion, 30 mg of oral risedronate administered daily for 84 days significantly reduced the biochemical indices of disease activity and was associated with pain reduction in patients with moderate to severe Paget's disease of bone. Normalization of ALP was observed in the majority of patients. Repeated administration of risedronate was shown to be beneficial. In general, risedronate was well tolerated and demonstrated a good safety profile.     

Structural and functional characterisation of two proteolytic fragments of the bacterial protein toxin, pneumolysin

Proteolytic cleavage of the bacterial protein toxin pneumolysin with protease K creates two fragments of 37 and 15 kDa. This paper describes the purification of these two fragments and their subsequent physical and biological characterisation. The larger fragment is directly involved in the cytolytic mechanism of this pore-forming protein, via membrane binding and self-association. The smaller fragment lacks ordered structure or discernible activity.     

Abnormal G protein alpha(s) - and alpha(i2)-subunit mRNA expression in bipolar affective disorder

Disturbances of events associated with intracellular signaling pathways have been suspected of involvement in the development or progression of affective disorders. Often, heterotrimeric G proteins are located at the beginning of these pathways as modulators of extracellular messages. For this reason, messenger RNA expression of three G protein alpha-subunits and of phosphatidylinositol-3 kinase (PI-3 K) regulatory subunit p85 was examined in granulocytes from patients with bipolar or unipolar affective disorder and compared to healthy controls. Messenger RNA expression of the G protein subunit alpha(q) and of p85 was identical in unipolar and bipolar patients and in controls. Furthermore, mRNAs of G protein subunits alpha(s) and alpha(i2) were not different in unipolar patients as compared to healthy controls. Alpha(s) mRNA, however, was markedly increased in bipolar patients. This increase was observed in lithium-treated (more than 12 months) and in unmedicated patients. Elevated levels of alpha(i2) mRNA in unmedicated bipolar patients did not reach statistical significance, whereas mRNA in bipolar patients receiving lithium was significantly above controls. Finally, long-term medication of unipolar patients with lithium had no influence on alpha(i2) mRNA levels. The data reveal elevated mRNA levels of G alpha(s) as a robust feature of bipolar affective disorder. Moreover, despite responsiveness of alpha(i2) gene expression to cAMP-related events, no substantial upregulation of alpha(i2) mRNA was observed in bipolar patients. The lack of alpha(i2) mRNA upregulation, hence, could be an additional abnormality in these patients. Even though lithium was able to reinstate this upregulation, there was no feedback downregulation of alpha(s). This strongly supports the notion of major disturbances of the cAMP signaling system in bipolar illness.     

Small nucleolar RNAs and pre-rRNA processing in plants

Transduction of MDR1 may be of use in chemoprotection of normal bone marrow (BM) cells during treatment of malignancies, or as a selectable marker for the transfer of other genes into the BM, a critical target for the cure of many diseases. To that aim, the human multidrug resistance gene MDR1 was cloned into an SV40 pseudoviral vector containing the SV40 origin of replication (ori) and encapsidation signal (ses), and the plasmid was encapsidated in COS cells as SV40/MDR1 pseudovirions. Expression of the human MDR1 gene was demonstrated in murine MEL cells infected with SV40/MDR1 pseudovirions, using a monoclonal antibody (MPK16) specific for the human 170-kD P-glycoprotein. Functional P-glycoprotein was demonstrated by resistance to colchicine in NIH-3T3 cells infected with SV40/MDR1 pseudovirions. Activity of P-glycoprotein was assayed by rhodamine-123 dye exclusion and fluorescence-activated cell sorter analysis (FACS) in various cell types including hematopoietic cells. Highly efficient gene transfer and expression was demonstrated in all murine and human cell types tested, including primary human BM cells. Using multiplicities of infection (moi) of 1-2, over 95% of cells were found to become MDR1+. The percent of MDR1+ cells was proportional to the moi. We conclude that the SV40 pseudoviral vector is efficient for gene transmission into human hematopoietic cells.     

Significant correlation between thrombospondin 1 and serine proteinase expression in rheumatoid synovium

OBJECTIVES: The natural history of Helicobacter pylori infection in humans is not well established. We aimed to systematically review the literature on the natural acquisition and spontaneous elimination of the infection and its clinical consequences. METHODS: A broad-based MEDLINE and Current Contents search was performed to identify all related publications between 1986 and 1996. Abstracts from recent major conferences that provided adequate data were also included. RESULTS: The prevalence of H. pylori infection increases with age, the rates being significantly lower in developed countries than in developing countries. However, the overall prevalence is decreasing in both developing and developed countries, which probably is responsible for the steep decline of gastric cancer in some industrialized countries. The natural acquisition of H. pylori infection occurs, for the most part, in childhood, and it appears that the incidence is currently slightly higher in developing countries than in industrialized countries. Spontaneous elimination of the infection also occurs, especially in young children and the elderly. The route of transmission continues to be uncertain, with the best evidence favoring both the gastro-oral and fecal-oral routes. Low socioeconomic status is a major risk factor for acquisition of the infection. Genetics probably plays a role in the acquisition or clearance of H. pylori infection in individuals. CONCLUSIONS: Low rates of natural acquisition and elimination of the infection in adults suggest that it is worthwhile to eradicate the organism from adults, but there should be further evaluation of the need for eradication of H. pylori in children.     

Validity and cost-effectiveness of antisperm antibody testing before in vitro fertilization

OBJECTIVE: To determine the usefulness of and cost-effectiveness of antisperm antibody testing in the prediction of poor fertilization rates in couples undergoing IVF. DESIGN: Retrospective cohort study. SETTING: A hospital-based reproductive endocrinology and infertility practice. PATIENT(S): Male partners of 251 couples undergoing IVF between 1992 and 1997. MAIN OUTCOME MEASURE(S): Fertilization rates in couples undergoing conventional IVF. RESULT(S): One hundred nineteen couples were evaluated for antisperm antibodies; fertilization rates were similar in those couples whose husbands were and were not tested (64% versus 68%). Antisperm antibodies were detected in 16 men. Four (25%) of the 16 couples whose husbands had antisperm antibodies fertilized < or = 50% of oocytes, compared with 31 (30%) of the 103 couples whose husbands did not have these antibodies. Overall, 21 couples (8.4%) experienced complete fertilization failure. In a program that included antisperm antibody testing for selected couples and intracytoplasmic sperm injection (ICSI) for those who tested positive, it would cost $11,735 to prevent a fertilization failure (assuming ICSI were 100% effective), whereas it would cost $9,250 to perform ICSI in a second IVF cycle for those who initially failed. CONCLUSION(S): In this practice setting, antisperm antibody testing has low sensitivity in predicting low or no fertilization and does not appear to be cost-effective when selectively ordered as part of an IVF workup.