A Drosophila homolog of bovine smg p25a GDP dissociation inhibitor undergoes a shift in isoelectric point in the developmental mutant quartet

The Drosophila developmental mutation quartet causes late larval lethality and small imaginal discs and, when expressed in the adult female, has a lethal effect on early embryogenesis. These developmental defects are associated with mitotic defects, which include a low mitotic index in larval brains and incomplete separation of chromosomes in mitosis in the early embryo. quartet mutations also have a biochemical effect, i.e., a basic shift in isoelectric point in three proteins. We have purified one of these proteins, raised an antibody to it, and isolated and sequenced its cDNA. At the amino acid level, the sequence shows 68% identity and 81% similarity to bovine smg p25a GDP dissociation inhibitor (GDI), a regulator of ras-like small GTPases of the rab/SEC4/YPT1 subfamily. The correlation between a basic shift in isoelectric point in Drosophila GDI in quartet mutant tissue and the quartet developmental phenotype raises the possibility that a posttranslational modification of GDI is necessary for its function and that GDI function is essential for development.     

Evolution of the serum amyloid A (SAA) protein superfamily

The serum amyloid A (SAA) superfamily comprises a number of genes and proteins characterized from a range of mammalian species. The majority of members described to date are dramatically induced during the acute-phase response, suggesting an important short-term beneficial role in the response to tissue injury and inflammation. However, important disease associations have also been proposed for certain SAAs during chronic inflammation. The nomenclature of many of the superfamily members has been the result of comparisons with previously reported sequences implying disease association and/or functional relatedness between such members. The evolutionary relationships of the SAA superfamily members have been investigated by comparisons at both the amino acid and the nucleotide level. The results indicate that all members of the superfamily within a species have been undergoing concerted evolution. This has important implications in ascribing functions and disease associations to individual SAA superfamily members and indicates that designations should not be based on the extent of amino acid identity alone but should be made only following direct experimental observation of the proteins themselves.     

Laboratory evaluation of the XR-Bond Dentin/Enamel Bonding System

The shear bond strengths of the XR-Bonding System used in conjunction with Herculite composite, to the dentine of forty extracted human permanent first and second molars were determined after the test specimens were stored in physiological saline at 37 degrees C for 48 hours, one week, two weeks and four weeks, respectively. A shear load was applied to the base of the bonded composite cylinders with a knife-edged rod at a crosshead speed of 0.5 mm/minute. The shear bond strengths were expressed in megapascals (MPa). The quantitative microleakage of Class V preparations in dentine (cementum) in forty-eight extracted human maxillary permanent canines restored with the same dentinal bonding system and after storage in physiological saline at 37 degrees C for the same time intervals as for the shear bond strength tests, was determined. On the final day of each time interval the teeth were thermocycled X 500 in a 2 per cent methylene blue solution between 8 degrees C and 50 degrees C with a dwell time of 15 seconds. Microleakage was determined by a spectrophotometric dye-recovery method and expressed in microgram dye/restoration. There was a significant trend for the shear bond strengths to increase with duration of storage (p = 0.01) but the quantitative microleakage was not significantly different (p = 0.75).     

Overview of randomized trials of intravenous heparin in patients with acute myocardial infarction treated with thrombolytic therapy

Intravenous heparin is routinely given after thrombolytic therapy for patients with acute myocardial infarction in the United States and in some, but by no means all, other countries. Several trials have documented improved infarct-artery patency in patients treated with heparin; however, none was large enough individually to assess the effect of heparin on clinical outcomes. We performed a systematic overview of the 6 randomized controlled trials (1,735 patients) to summarize the available data concerning the risks and benefits of intravenous heparin versus no heparin after thrombolytic therapy. Mortality before hospital discharge was 5.1% for patients allocated to intravenous heparin compared with 5.6% for controls (relative risk reduction of 9%, odds ratio 0.91, 95% confidence interval 0.59 to 1.39). Similar rates of recurrent ischemia and reinfarction were observed among those allocated to heparin therapy or control. The rates of total stroke, intracranial hemorrhage, and severe bleeding were similar in patients allocated to heparin; however, the risk of any severity of bleeding was significantly higher (22.7% vs 16.2%; odds ratio 1.55, 95% confidence interval 1.21 to 1.98). There was no significant difference in the observed effects of heparin between patients receiving tissue-type plasminogen activator and those receiving streptokinase or anisoylated plasminogen streptokinase activator complex, or between patients who did and did not receive aspirin. The findings of this overview demonstrate that insufficient clinical outcome data are available to support or to refute the routine use of intravenous heparin therapy after thrombolysis. It is not known if these findings are due to lack of statistical power, inappropriate levels of anticoagulation, or lack of benefit of intravenous heparin. Large randomized studies of heparin (and of new antithrombotic regimens) are needed to establish the role of such therapy.     

Expression of 72-kDa gelatinase (matrix metalloproteinase-2) in the developing mouse craniofacial complex

Tissue remodelling is an important feature during embryogenesis. Although the matrix metalloproteinases are believed to participate in these processes, the relation between matrix metalloproteinases and tissue remodelling during craniofacial morphogenesis remains unclear. The purpose of the study was to look for the presence of enzymes involved in extracellular matrix degradation during craniofacial morphogenesis. Protein expression of the matrix metalloproteinase, 72-kDa gelatinase (matrix metalloproteinase-2, gelatinase A, 72-kDa type IV collagenase) was studied by gelatine zymography and by indirect immunofluorescence with conventional and confocal microscopy. In the anterior region of the developing mouse face, 72-kDa gelatinase was labelled mainly in the tips and peripheral regions of the nasal and facial prominences. Upon contact and fusion of the prominences, the staining was intensely localized to the zone of the fusion and the tips and peripheral regions of the nasal prominences and the maxilla. The labelling of 72-kDa gelatinase was also present in the peripheral regions of the mandible, second branchial arch, and the face around the developing eye. However, during lens vesicle formation, the staining of 72-kDa gelatinase was absent in the invaginated lens ectoderm. After the lens had completely detached from the surface ectoderm, the staining was resumed in the corneal epithelium and mesenchyme. Gelatine zymography was used to confirm the presence of active and latent 72-kDa gelatinase in the developing mouse craniofacial complex. Collectively, these data indicate that 72-kDa gelatinase may play a significant part in localized tissue remodelling during craniofacial morphogenesis and the aberrant expression or function of the enzyme could be involved in causing facial abnormalities.     

Abrupt (< 1 day), acute (< 1 week), and early (< 1 month) vessel closure at the angioplasty site. Morphologic observations and causes of closure in 130 necropsy patients undergoing coronary angioplasty

While abundant clinical and angiographic data are available regarding features of acute or abrupt closure at the site of balloon angioplasty, little morphologic information is available. This study discusses morphologic-histologic causes for acute closure after angioplasty in 130 necropsy patients. Intimal-medial flaps, elastic recoil, and primary thrombosis were the three leading morphologic causes for closure. Data were subdivided into time categories: abrupt (< 1 day), acute (< 1 week), and early (< 1 month). Intimal-medial flaps remained the most common cause for angioplasty closure despite time from angioplasty to documented occlusion. Morphologic recognition of types and frequencies of angioplasty closure are discussed, and specific mechanical, pharmacologic, or combined treatments are reviewed.     

Purification and characterization of an alpha1,2,-L-fucosyltransferase, which modifies the cytosolic protein FP21,from the cytosol of Dictyostelium

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.